DR1: Mol Cell Biol. 2003 Dec;23(23):8902-12. 

Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a
p53-dependent ribosomal-stress checkpoint pathway.

Zhang Y, Wolf GW, Bhat K, Jin A, Allio T, Burkhart WA, Xiong Y.

Department of Molecular and Cellular Oncology, The University of Texas M. D.
Anderson Cancer Center, Houston, Texas 77030, USA. ypzhang@mdanderson.org

The gene encoding p53 mediates a major tumor suppression pathway that is
frequently altered in human cancers. p53 function is kept at a low level during
normal cell growth and is activated in response to various cellular stresses.
The MDM2 oncoprotein plays a key role in negatively regulating p53 activity by
either direct repression of p53 transactivation activity in the nucleus or
promotion of p53 degradation in the cytoplasm. DNA damage and oncogenic insults,
the two best-characterized p53-dependent checkpoint pathways, both activate p53
through inhibition of MDM2. Here we report that the human homologue of MDM2,
HDM2, binds to ribosomal protein L11. L11 binds a central region in HDM2 that is
distinct from the ARF binding site. We show that the functional consequence of
L11-HDM2 association, like that with ARF, results in the prevention of
HDM2-mediated p53 ubiquitination and degradation, subsequently restoring
p53-mediated transactivation, accumulating p21 protein levels, and inducing a
p53-dependent cell cycle arrest by canceling the inhibitory function of HDM2.
Interference with ribosomal biogenesis by a low concentration of actinomycin D
is associated with an increased L11-HDM2 interaction and subsequent p53
stabilization. We suggest that L11 functions as a negative regulator of HDM2 and
that there might exist in vivo an L11-HDM2-p53 pathway for monitoring ribosomal
integrity.

PMID: 14612427 [PubMed - indexed for MEDLINE]



NR2: Exp Parasitol. 2003 Jul-Aug;104(3-4):113-21. 

Ascaris suum: cDNA microarray analysis of 4th stage larvae (L4) during self-cure
from the intestine.

Morimoto M, Zarlenga D, Beard H, Alkharouf N, Matthews BF, Urban JF Jr.

United States Department of Agriculture, Agricultural Research Service, Nutrient
Requirements and Functions Laboratory, Beltsville Human Nutrition Research
Center, Beltsville, MD 20705, USA.

There is spontaneous cure of a large portion of Ascaris suum 4th-stage larvae
(L4) from the jejunum of infected pigs between 14 and 21 days after inoculation
(DAI). Those L4 that remain in the jejunum continue to develop while those that
have moved to the ileum are eventually expelled from the intestines. Although
increases in intestinal mucosal mast cells and changes in local host immunity
are coincidental with spontaneous cure, the population of L4 that continue to
develop in the jejunum may counteract host protective mechanisms by the
differential production of factors related to parasitism. To this end, a cDNA
library was constructed from L4 isolated from pig jejunum at 21 DAI, and 93% of
1920 original clones containing a single amplicon in the range 400-1500 bp were
verified by gel electrophoresis and printed onto glass slides for microarray
analysis. Fluorescent probes were prepared from total RNA isolated from: (1) 3rd
stage-larvae from lung at 7 DAI, (L3); (2) L4 from jejunum at 14 DAI (L4-14-J);
(3) L4 from jejunum at 21 DAI (L4-21-J); (4) L4 from ileum at 21 DAI (L4-21-I,
and; (5) adults (L5). Cy3-labeled L3, L4-14-J, L4-21-I and L5 cDNA, and
Cy5-labeled L4-21-J cDNA were simultaneously used to screen the printed arrays
containing the L4-21-J-derived cDNA library. Several clones showed consistent
differential gene expression over two separate experiments and were grouped into
3 distinct transcription patterns. The data showed that sequences from muscle
actin and myosin, ribosomal protein L11, glyceraldehyde-3-phosphate
dehydrogenase and the flavoprotein subunit of succinate dehydrogenase were
highly expressed in L4-21-J, but not in L4-21-I; as were a collection of
un-annotated genes derived from a worm body wall-hypodermis library, and a
testes germinal zone tissue library. These results suggest that only actively
developing A. suum L4 are destined to parasitize the host and successfully
neutralize host protective responses.

PMID: 14552858 [PubMed - indexed for MEDLINE]



NR3: J Protein Chem. 2003 Apr;22(3):249-58. 

Characterization and analysis of posttranslational modifications of the human
large cytoplasmic ribosomal subunit proteins by mass spectrometry and Edman
sequencing.

Odintsova TI, Muller EC, Ivanov AV, Egorov TA, Bienert R, Vladimirov SN, Kostka
S, Otto A, Wittmann-Liebold B, Karpova GG.

Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow,
Russian Federation.

The 60S ribosomal proteins were isolated from ribosomes of human placenta and
separated by reversed phase HPLC. The fractions obtained were subjected to
trypsin and Glu-C digestion and analyzed by mass fingerprinting (MALDI-TOF),
MS/MS (ESI), and Edman sequencing. Forty-six large subunit proteins were found,
22 of which showed masses in accordance with the SwissProt database (June 2002)
masses (proteins L6, L7, L9, L13, L15, L17, L18, L21, L22, L24, L26, L27, L30,
L32, L34, L35, L36, L37, L37A, L38, L39, L41). Eleven (proteins L7, L10A, L11,
L12, L13A, L23, L23A, L27A, L28, L29, and P0) resulted in mass changes that are
consistent with N-terminal loss of methionine, acetylation, internal
methylation, or hydroxylation. A loss of methionine without acetylation was
found for protein L8 and L17. For nine proteins (L3, L4, L5, L7A, L10, L14, L19,
L31, and L40), the molecular masses could not be determined. Proteins P1 and
protein L3-like were not identified by the methods applied.

PMID: 12962325 [PubMed - indexed for MEDLINE]



NR4: Chem Biol. 2003 Aug;10(8):769-78. 

Structural basis for contrasting activities of ribosome binding thiazole
antibiotics.

Lentzen G, Klinck R, Matassova N, Aboul-ela F, Murchie AI.

RiboTargets, Ltd., Granta Park, Abington, CB1 6GB, Cambridge, United Kingdom.

Thiostrepton and micrococcin inhibit protein synthesis by binding to the L11
binding domain (L11BD) of 23S ribosomal RNA. The two compounds are structurally
related, yet they produce different effects on ribosomal RNA in footprinting
experiments and on elongation factor-G (EF-G)-dependent GTP hydrolysis. Using
NMR and an assay based on A1067 methylation by thiostrepton-resistance
methyltransferase, we show that the related thiazoles, nosiheptide and siomycin,
also bind to this region. The effect of all four antibiotics on EF-G-dependent
GTP hydrolysis and EF-G-GDP-ribosome complex formation was studied. Our NMR and
biochemical data demonstrate that thiostrepton, nosiheptide, and siomycin share
a common profile, which differs from that of micrococcin. We have generated a
three-dimensional (3D) model for the interaction of thiostrepton with L11BD RNA.
The model rationalizes the differences between micrococcin and the
thiostrepton-like antibiotics interacting with L11BD.

PMID: 12954336 [PubMed - indexed for MEDLINE]



NR5: Bioorg Med Chem Lett. 2003 Aug 4;13(15):2455-8. 

Structure-based design of agents targeting the bacterial ribosome.

Bower J, Drysdale M, Hebdon R, Jordan A, Lentzen G, Matassova N, Murchie A,
Powles J, Roughley S.

Department of Medicinal Chemistry, RiboTargets Ltd., Granta Park, Abington,
Cambridge CB1 6GB, UK.

Rational structure-based drug design has been applied to the antibiotic
thiostrepton, in an attempt to overcome some of its' limitations. The
identification of a proposed binding fragment allowed construction of a number
of key fragments, which were derivatised to generate a library of potential
antibiotics. These were then evaluated to determine their ability to bind to the
L11 binding domain of the prokaryotic ribosome and inhibit bacterial protein
translation.

PMID: 12852942 [PubMed - indexed for MEDLINE]



DR6: Cancer Cell. 2003 Jun;3(6):577-87. 

Regulation of HDM2 activity by the ribosomal protein L11.

Lohrum MA, Ludwig RL, Kubbutat MH, Hanlon M, Vousden KH.

Regulation of Cell Growth Laboratory, NCI-FRCDC, Frederick, MD 21702, USA.

The HDM2 protein plays an important role in regulating the stability and
function of the p53 tumor suppressor protein. In this report, we show that the
ribosomal protein L11 can interact with HDM2 and inhibit HDM2 function, thus
leading to the stabilization and activation of p53. The inhibition of HDM2
activity by L11 shows some similarity to the previously described activity of
ARF, and expression of either ARF or L11 can induce a p53 response. Enhancement
of the interaction between endogenous L11 and HDM2 following treatment of cells
with low levels of actinomycin-D suggests that the HDM2/L11 interaction
represents a novel pathway for p53 stabilization in response to perturbations in
ribosome biogenesis.

PMID: 12842086 [PubMed - indexed for MEDLINE]



NR7: Mol Biol Rep. 2003 Jun;30(2):113-9. 

Specific binding of ribosome recycling factor (RRF) with the Escherichia coli
ribosomes by BIACORE.

Todorova RT, Saihara Y.

Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.
todorova@obzor.bio21.bas.bg

The direct assays on Biacore with immobilised RRF and purified L11 from E. coli
in the flow trough have shown unspecific binding between the both proteins. The
interaction of RRF with GTPase domain of E. coli ribosomes, a functionally
active complex of L11 with 23S r RNA and L10.(L7/L12)4 was studied by Biacore.
In the experiments of binding of RRF with 30S, 50S and 70S ribosomes from E.
coli were used the antibiotics thiostrepton, tetracycline and neomycin and
factors, influencing the 70S dissociation Mg2+, NH4Cl, EDTA. The binding is
strongly dependent from the concentrations of RRF, Mg2+, NH4Cl, EDTA and is
inhibited by thiostrepton. The effect is most specific for 50S subunits and
indicates that the GTPase centre can be considered as a possible site of
interaction of RRF with the ribosome. We can consider an electrostatic character
of the interactions with most probable candidate 16S and 23S r RNA at the
interface of 30S and 50S ribosomal subunits.

PMID: 12841582 [PubMed - indexed for MEDLINE]



NR8: J Mol Biol. 2003 Jun 27;330(1):9-13. 

Site of functional interaction of release factor 1 with the ribosome.

Van Dyke N, Murgola EJ.

Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer
Center, Box 11, 1515 Holcombe Boulevard, Houston 77030-4009, USA.

Ribosomal protein L11 consists of a C-terminal and an N-terminal domain. To
determine the importance of each domain for interaction with release factor 1,
which works specifically at the UAG termination codon, we constructed
Escherichia coli strains lacking either the entire L11 protein or just the
N-terminal portion. Strains lacking L11 exhibited UAG suppression, defective
growth, and high-temperature lethality, phenotypes that were reversed by
expression of L11 protein from a plasmid. Strains lacking only the N-terminal
portion of L11 grew well at physiological temperatures and survived at high
temperature, but they were defective in UAG-dependent termination. Our results
show for the first time that it is precisely the N-terminal part of ribosomal
protein L11 that is required for the functional interaction of release factor 1
with the ribosome in the cell.

PMID: 12818198 [PubMed - indexed for MEDLINE]



NR9: Mol Biol (Mosk). 2003 May-Jun;37(3):425-35. 

[Structural and functional analysis of the human ribosomal protein L11 gene]

[Article in Russian]

Voronina EN, Kolokol'tsova TD, Nechaeva EA, Filipenko ML.

Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian
Academy of Sciences, Novosibirsk, 630090 Russia. voronina_l@yahoo.com

The nucleotide sequence was established for the human ribosomal protein L11 gene
(HRPL11). The gene (4583 bp) proved to consist of six exons and five introns.
The structure of the HRPL11 promoter was shown to be typical for ribosomal
protein genes of higher eukaryotes: the promoter lacks TATA or CAAT boxes and
has a TATA-like element (ATAA, -24 ... -27) surrounded by GC-rich regions, the
transcription start point is within an oligopyrimidine tract, and the
5'-untranslater region is short (26 bp). Proteins interacting with the HRPL11
promoter were identified by the electrophoretic mobility shift assay with
oligonucleotide competitors containing known transcription factor-binding sites.
The promoter regions were compared for the human genes for ribosomal proteins
L11, L32, and S26.

PMID: 12815950 [PubMed - indexed for MEDLINE]



NR10: Acta Crystallogr D Biol Crystallogr. 2003 May;59(Pt 5):930-2. Epub 2003 Apr
25. 

Crystallization and preliminary X-ray diffraction analysis of ribosomal protein
L11 methyltransferase from Thermus thermophilus HB8.

Kaminishi T, Sakai H, Takemoto-Hori C, Terada T, Nakagawa N, Maoka N, Kuramitsu
S, Shirouzu M, Yokoyama S.

RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045,
Japan.

Ribosomal proteins are subjected to a variety of post-translational
modifications, of which methylation is the most frequently found in all three
kingdoms of life. PrmA is the only bacterial enzyme identified to date that
catalyzes the methylation of a ribosomal protein. It is responsible for the
introduction of nine methyl groups into the N-terminal domain of ribosomal
protein L11. The PrmA protein from Thermus thermophilus HB8 was crystallized and
a preliminary X-ray diffraction analysis was performed. A cryocooled crystal
diffracted X-rays beyond 1.9 A using synchrotron radiation.

PMID: 12777815 [PubMed - indexed for MEDLINE]



NR11: J Biomol NMR. 2003 Feb;25(2):163-4. 

NMR assignment of the full-length ribosomal protein L11 from Thermotoga
maritima.

Ilin S, Hoskins A, Schwalbe H, Wohnert J.

Publication Types:
    Letter

PMID: 12652127 [PubMed - indexed for MEDLINE]



NR12: EMBO J. 2003 Feb 17;22(4):945-53. 

Mechanism of Tet(O)-mediated tetracycline resistance.

Connell SR, Trieber CA, Dinos GP, Einfeldt E, Taylor DE, Nierhaus KH.

Department of Medical Microbiology and Immunology, University of Alberta,
Edmonton, AB, T6G 2H7, Canada.

Tet(O) is an elongation factor-like protein which confers resistance to the
protein synthesis inhibitor tetracycline by promoting the release of the drug
from its inhibitory site on the ribosome. Here we investigated the interaction
of Tet(O) with the elongating ribosome and show, using dimethyl sulfate (DMS)
probing and binding assays, that it interacts preferentially with the
post-translocational ribosome. Furthermore, using an XTP-dependent mutant of
Tet(O), we demonstrated that Tet(O) induces conformational rearrangements within
the ribosome which can be detected by EF-Tu, and manifested as a stimulation in
the GTPase activity of this elongation factor. As such, these conformational
changes probably involve the ribosomal GTPase-associated center and,
accordingly, Tet(O) alters the DMS modification pattern of the L11 region.
Additionally, tetracycline binding is associated with an E(a) of 58 kJ/mol.
These results suggest a model where both Tet(O) and tetracycline induce a
conformational change in functionally opposite directions and the Tet(O)-induced
conformation persists after it has left the ribosome; this prevents rebinding of
the drug while allowing productive A-site occupation by a ternary complex in the
presence of tetracycline.

PMID: 12574130 [PubMed - indexed for MEDLINE]



NR13: Microbiology. 2002 Nov;148(Pt 11):3365-73. 

Genetic and physiological characterization of rpoB mutations that activate
antibiotic production in Streptomyces lividans.

Lai C, Xu J, Tozawa Y, Okamoto-Hosoya Y, Yao X, Ochi K.

National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642,
Japan.

Antibiotic production in Streptomyces lividans can be activated by introducing
certain mutations (rif) into the rpoB gene that confer resistance to rifampicin.
Working with the most typical (rif-17) mutant strain, KO-417, the rif-17
mutation was characterized. The rif-17 mutation was shown to be responsible for
activating antibiotic production and for reducing the growth rate of strain
KO-417, as demonstrated by gene-replacement experiments. Gene-expression
analysis revealed that introduction of rif into S. lividans elevates expression
of the pathway-specific regulatory gene actII-ORF4 to nearly the same level seen
in Streptomyces coelicolor. The rif effect on antibiotic production was still
evident in the genetic background of relC, indicating that the rif mutation can
provoke its effect without depending on ppGpp. Accompanying the restoration of
antibiotic production, rif mutants also exhibited a lower rate of RNA synthesis
compared to the parental strain when grown in a nutritionally rich medium,
suggesting that the mutant RNA polymerases may behave like 'stringent' RNA
polymerases. These results indicate that the rif mutation can alter the
gene-expression pattern independent of ppGpp. The impaired growth of strain
KO-417 (rif-17) was largely restored by introducing the second rif mutation
(rif-18) just adjacent to the rif-17 position. Proteome analysis using
two-dimensional PAGE revealed that the rif mutant strain KO-418 (rif-17 rif-18)
displayed a temporal burst of expression especially of two enzymes, glutamine
synthetase (type II) and oxidoreductase, during the late growth phase.

PMID: 12427928 [PubMed - indexed for MEDLINE]



NR14: Mol Cell. 2002 Oct;10(4):779-88. 

Dissection of the mechanism for the stringent factor RelA.

Wendrich TM, Blaha G, Wilson DN, Marahiel MA, Nierhaus KH.

Fachbereich Chemie-Biochemie, Philipps-Universitat Marburg,
Hans-Meerwein-Strasse, D-35032, Marburg, Germany.

During conditions of nutrient deprivation, ribosomes are blocked by uncharged
tRNA at the A site. The stringent factor RelA binds to blocked ribosomes and
catalyzes synthesis of (p)ppGpp, a secondary messenger that induces the
stringent response. We demonstrate that binding of RelA and (p)ppGpp synthesis
are inversely coupled, i.e., (p)ppGpp synthesis decreases the affinity of RelA
for the ribosome. RelA binding to ribosomes is governed primarily by mRNA, but
independently of ribosomal protein L11, while (p)ppGpp synthesis strictly
requires uncharged tRNA at the A site and the presence of L11. A model is
proposed whereby RelA hops between blocked ribosomes, providing an explanation
for how low intracellular concentrations of RelA (1/200 ribosomes) can
synthesize (p)ppGpp at levels that accurately reflect the starved ribosome
population.

PMID: 12419222 [PubMed - indexed for MEDLINE]



NR15: J Biol Chem. 2003 Jan 10;278(2):1259-67. Epub 2002 Oct 29. 

Dissociation of intact Escherichia coli ribosomes in a mass spectrometer.
Evidence for conformational change in a ribosome elongation factor G complex.

Hanson CL, Fucini P, Ilag LL, Nierhaus KH, Robinson CV.

Department of Chemistry, University of Cambridge, Lensfield Road, United
Kingdom.

We used mass spectrometry to identify proteins that are released in the gas
phase from Escherichia coli ribosomes in response to a range of different
solution conditions and cofactor binding. From solution at neutral pH the
spectra are dominated by just 4 of the 54 ribosomal proteins (L7/L12, L11, and
L10). Lowering the pH of the solution leads to the gas phase dissociation of
four additional proteins as well as the 5 S RNA. Replacement of Mg(2+) by Li(+)
ions in solutions of ribosomes induced the dissociation of 17 ribosomal
proteins. Correlation of these results with available structural information for
ribosomes revealed that a relatively high interaction surface area of the
protein with RNA was the major force in preventing dissociation. By using the
proteins that dissociate to probe their interactions with RNA, we examined
different complexes of the ribosome formed with the elongation factor G and
inhibited by fusidic acid or thiostrepton. Mass spectra recorded for the fusidic
acid-inhibited complex reveal subtle changes in peak intensity of the proteins
that dissociate. By contrast gas phase dissociation from the
thiostrepton-inhibited complex is markedly different and demonstrates the
presence of L5 and L18, two proteins that interact exclusively with the 5 S RNA.
These results allow us to propose that the ribosome elongation factor-G complex
inhibited by thiostrepton, but not fusidic acid, involves destabilization of 5 S
RNA-protein interactions.

PMID: 12409297 [PubMed - indexed for MEDLINE]



NR16: Bioorg Chem. 2002 Jun;30(3):163-87. 

Large-scale motions within ribosomal 50S subunits as demonstrated using
photolabile oligonucleotides.

Seo HS, Cooperman BS.

Department of Chemistry, University of Pennsylvania, Philadelphia, 19104, USA.

Photolabile oligonucleotides (PHONTs) bind to rRNA sequences to which they are
complementary and, on photolysis, incorporate into neighboring ribosomal
components. Here we report on photocrosslinking results obtained with PHONTs
targeting 23S rRNA nucleotides 1882-1892, in the long lateral arm of the 50S
subunit (PHONT 1892), and 1085-1093, in the L11 binding domain (PHONT 1093).
Photolysis of the PHONT 1892.50S and PHONT 1093.50S complexes leads to formation
of 'long-range' crosslinks from C1892 to U1094/A1095 and G1950, and from G1093
to U1712/1716 and U1926, that are clearly incompatible with published crystal
structures of 50S subunits. These results provide strong evidence that within
the 50S subunit (a) the L11 binding domain can extend in an arm-like fashion,
accessing large areas of the ribosome, and (b) the lateral arm can bend about
the noncanonical helix at its center. Such motions may have functional relevance
in identifying regions that undergo major conformational change as the ribosome
moves through its catalytic cycle.

PMID: 12406702 [PubMed - indexed for MEDLINE]



NR17: J Bacteriol. 2002 Nov;184(22):6155-62. 

Proteins released by Helicobacter pylori in vitro.

Kim N, Weeks DL, Shin JM, Scott DR, Young MK, Sachs G.

Department of Physiology and Medicine, UCLA Digestive Research Center,
University of California, Los Angeles, and VA Greater Los Angeles Health Care
System, 90073, USA.

Secretion of proteins by Helicobacter pylori may contribute to gastric
inflammation and epithelial damage. An in vitro analysis was designed to
identify proteins released by mechanisms other than nonspecific lysis. The
radioactivity of proteins in the supernatant was compared with that of the
intact organism by two-dimensional gel phosphorimaging following a 4-h
pulse-chase. The ratio of the amount of UreB, a known cytoplasmic protein, in
the supernatant to that in the pellet was found to be 0.25, and this was taken
as an index of lysis during the experiments (n = 6). Ratios greater than that of
UreB were used to distinguish proteins that were selectively released into the
medium. Thus, proteins enriched more than 10-fold in the supernatant compared to
UreB were identified by mass spectrometry. Sixteen such proteins were present in
the supernatant: VacA; a conserved secreted protein (HP1286); putative peptidyl
cis-trans isomerase (HP0175); six proteins encoded by HP0305, HP0231, HP0973,
HP0721, HP0129, and HP0902; thioredoxin (HP1458); single-stranded-DNA-binding
12RNP2 precursor (HP0827); histone-like DNA-binding protein HU (HP0835);
ribosomal protein L11 (HP1202); a putative outer membrane protein (HP1564); and
outer membrane proteins Omp21 (HP0913) and Omp20 (HP0912). All except HP0902,
thioredoxin, HP0827, HP0835, and HP1202 had a signal peptide. When nalidixic
acid, a DNA synthesis inhibitor, was added to inhibit cell division but not
protein synthesis, to decrease possible contamination due to outer membrane
shedding, two outer membrane proteins (Omp21 and Omp20) disappeared from the
supernatant, and the amount of VacA also decreased. Thus, 13 proteins were still
enriched greater than 10-fold in the medium after nalidixic acid treatment,
suggesting these were released specifically, possibly by secretion. These
proteins may be implicated in H. pylori-induced effects on the gastric
epithelium.

Publication Types:
    Evaluation Studies

PMID: 12399485 [PubMed - indexed for MEDLINE]



NR18: Nat Struct Biol. 2002 Nov;9(11):849-54. 

Ribosome interactions of aminoacyl-tRNA and elongation factor Tu in the
codon-recognition complex.

Stark H, Rodnina MV, Wieden HJ, Zemlin F, Wintermeyer W, van Heel M.

Max-Planck Institute for Biophysical Chemistry, 37077 Gottingen, Germany.
holger.stark@mpibpc.mpg.de

The mRNA codon in the ribosomal A-site is recognized by aminoacyl-tRNA (aa-tRNA)
in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here we report
the 13 A resolution three-dimensional reconstruction determined by cryo-electron
microscopy of the kirromycin-stalled codon-recognition complex. The structure of
the ternary complex is distorted by binding of the tRNA anticodon arm in the
decoding center. The aa-tRNA interacts with 16S rRNA, helix 69 of 23S rRNA and
proteins S12 and L11, while the sarcin-ricin loop of 23S rRNA contacts domain 1
of EF-Tu near the nucleotide-binding pocket. These results provide a detailed
snapshot view of an important functional state of the ribosome and suggest
mechanisms of decoding and GTPase activation.

PMID: 12379845 [PubMed - indexed for MEDLINE]



NR19: Biochemistry. 2002 Oct 15;41(41):12520-8. 

GTPase activation of elongation factors Tu and G on the ribosome.

Mohr D, Wintermeyer W, Rodnina MV.

Institute of Physical Biochemistry, University of Witten/Herdecke, Stockumer
Strasse 10, D-58448 Witten, Germany.

The GTPase activity of elongation factors Tu and G is stimulated by the
ribosome. The factor binding site is located on the 50S ribosomal subunit and
comprises proteins L7/12, L10, L11, the L11-binding region of 23S rRNA, and the
sarcin-ricin loop of 23S rRNA. The role of these ribosomal elements in factor
binding, GTPase activation, or functions in tRNA binding and translocation, and
their relative contributions, is not known. By comparing ribosomes depleted of
L7/12 and reconstituted ribosomes, we show that, for both factors, interactions
with L7/12 and with other ribosomal residues contribute about equally and
additively to GTPase activation, resulting in an overall 10(7)-fold stimulation.
Removal of L7/12 has little effect on factor binding to the ribosome. Effects on
other factor-dependent functions, i.e., A-site binding of aminoacyl-tRNA and
translocation, are fully explained by the inhibition of GTP hydrolysis. Based on
these results, we propose that L7/12 stimulates the GTPase activity of both
factors by inducing the catalytically active conformation of the G domain. This
effect appears to be augmented by interactions of other structural elements of
the large ribosomal subunit with the switch regions of the factors.

PMID: 12369843 [PubMed - indexed for MEDLINE]



NR20: Genes Dev. 2002 Oct 1;16(19):2497-508. 

A Drosophila fragile X protein interacts with components of RNAi and ribosomal
proteins.

Ishizuka A, Siomi MC, Siomi H.

Institute for Genome Research, Graduate School of Nutrition, University of
Tokushima, Tokushima 770-8503, Japan.

Fragile X syndrome is a common form of inherited mental retardation caused by
the loss of FMR1 expression. The FMR1 gene encodes an RNA-binding protein that
associates with translating ribosomes and acts as a negative translational
regulator. In Drosophila, the fly homolog of the FMR1 protein (dFMR1) binds to
and represses the translation of an mRNA encoding of the microtuble-associated
protein Futsch. We have isolated a dFMR1-associated complex that includes two
ribosomal proteins, L5 and L11, along with 5S RNA. The dFMR1 complex also
contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68).
AGO2 is an essential component for the RNA-induced silencing complex (RISC), a
sequence-specific nuclease complex that mediates RNA interference (RNAi) in
Drosophila. We show that Dmp68 is also required for efficient RNAi. We further
show that dFMR1 is associated with Dicer, another essential component of the
RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that dFMR1 is part of
the RNAi-related apparatus. Our findings suggest a model in which the RNAi and
dFMR1-mediated translational control pathways intersect in Drosophila. Our
findings also raise the possibility that defects in an RNAi-related machinery
may cause human disease.

PMID: 12368261 [PubMed - indexed for MEDLINE]



NR21: J Biol Chem. 2002 Nov 1;277(44):41401-9. Epub 2002 Aug 26. 

Ribosomal proteins at the stalk region modulate functional rRNA structures in
the GTPase center.

Uchiumi T, Honma S, Endo Y, Hachimori A.

Institute of High Polymer Research, Faculty of Textile Science and Technology,
Shinshu University, Ueda 386-8567, Japan. uchiumi@giptc.shinshu-u.ac.jp

Replacement of the L10.L7/L12 protein complex and L11 in Escherichia coli
ribosomes with the respective rat counterparts P0.P1/P2 and eukaryotic L12
causes conversion of ribosomal specificity for elongation factors from
prokaryotic elongation factor (EF)-Tu/EF-G to eukaryotic EF (eEF)-1alpha/eEF-2.
Here we have investigated the effects of protein replacement on the structure
and function of two rRNA domains around positions 1070 and 2660 (sarcin/ricin
loop) of 23 S rRNA. Protein replacement at the 1070 region in E. coli 50 S
subunits was demonstrated by chemical probing analysis. Binding of rat proteins
to the 1070 region caused increased accessibility of the 2660 and 1070 regions
to ligands for eukaryotic ribosomes: the ribotoxin pepocin for the 2660 region
(E. coli numbering), anti-28 S autoantibody for the 1070 region, and eEF-2 for
both regions. Moreover, binding of the E. coli L10.L7/L12 complex and L11 to the
1070 region was shown to be responsible for E. coli ribosomal accessibility to
another ribotoxin, gypsophilin. Ribosomal proteins at the 1070 region appear to
modulate the structures and functions of the 2660 and 1070 RNA regions in
slightly different modes in prokaryotes and eukaryotes.

PMID: 12198134 [PubMed - indexed for MEDLINE]



NR22: J Mol Biol. 2002 Aug 9;321(2):215-34. 

Modeling a minimal ribosome based on comparative sequence analysis.

Mears JA, Cannone JJ, Stagg SM, Gutell RR, Agrawal RK, Harvey SC.

Department of Biochemistry and Molecular Genetics, University of Alabama at
Birmingham, Birmingham, AL 35295-0005, USA.

We have determined the three-dimensional organization of ribosomal RNAs and
proteins essential for minimal ribosome function. Comparative sequence analysis
identifies regions of the ribosome that have been evolutionarily conserved, and
the spatial organization of conserved domains is determined by mapping these
onto structures of the 30S and 50S subunits determined by X-ray crystallography.
Several functional domains of the ribosome are conserved in their
three-dimensional organization in the Archaea, Bacteria, Eucaryotic nuclear,
mitochondria and chloroplast ribosomes. In contrast, other regions from both
subunits have shifted their position in three-dimensional space during
evolution, including the L11 binding domain and the alpha-sarcin-ricin loop
(SRL). We examined conserved bridge interactions between the two ribosomal
subunits, giving an indication of which contacts are more significant. The tRNA
contacts that are conserved were also determined, highlighting functional
interactions as the tRNA moves through the ribosome during protein synthesis. To
augment these studies of a large collection of comparative structural models
sampled from all major branches on the phylogenetic tree, Caenorhabditis elegans
mitochondrial rRNA is considered individually because it is among the smallest
rRNA sequences known. The C.elegans model supports the large collection of
comparative structure models while providing insight into the evolution of
mitochondrial ribosomes.

PMID: 12144780 [PubMed - indexed for MEDLINE]



NR23: Nucleic Acids Res. 2002 Jun 15;30(12):2620-7. 

Interaction among silkworm ribosomal proteins P1, P2 and P0 required for
functional protein binding to the GTPase-associated domain of 28S rRNA.

Shimizu T, Nakagaki M, Nishi Y, Kobayashi Y, Hachimori A, Uchiumi T.

Institute of High Polymer Research and Department of Applied Biological Science,
Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567,
Japan.

Acidic ribosomal phosphoproteins P0, P1 and P2 were isolated in soluble form
from silkworm ribosomes and tested for their interactions with each other and
with RNA fragments corresponding to the GTPase-associated domain of residues
1030-1127 (Escherichia coli numbering) in silkworm 28S rRNA in vitro. Mixing of
P1 and P2 formed the P1-P2 heterodimer, as demonstrated by gel mobility shift
and chemical crosslinking. This heterodimer, but neither P1 or P2 alone, tightly
bound to P0 and formed a pentameric complex, presumably as P0(P1-P2)2, assumed
from its molecular weight derived from sedimentation analysis. Complex formation
strongly stimulated binding of P0 to the GTPase-associated RNA domain. The
protein complex and eL12 (E.coli L11-type), which cross-bound to the E.coli
equivalent RNA domain, were tested for their function by replacing with the
E.coli counterparts L10.L7/L12 complex and L11 on the rRNA domain within the 50S
subunits. Both P1 and P2, together with P0 and eL12, were required to activate
ribosomes in polyphenylalanine synthesis dependent on eucaryotic elongation
factors as well as eEF-2-dependent GTPase activity. The results suggest that
formation of the P1-P2 heterodimer is required for subsequent formation of the
P0(P1-P2)2 complex and its functional rRNA binding in silkworm ribosomes.

PMID: 12060678 [PubMed - indexed for MEDLINE]



NR24: J Mol Biol. 2002 May 10;318(4):963-73. 

A compact RNA tertiary structure contains a buried backbone-K+ complex.

Conn GL, Gittis AG, Lattman EE, Misra VK, Draper DE.

Department of Chemistry, The Johns Hopkins University, 3400 North Charles
Street, Baltimore, MD 21218, USA.

The structure of a 58 nucleotide ribosomal RNA fragment buries several phosphate
groups of a hairpin loop within a large tertiary core. During refinement of an
X-ray crystal structure containing this RNA, a potassium ion was found to be
contacted by six oxygen atoms from the buried phosphate groups; the ion is
contained completely within the solvent-accessible surface of the RNA. The
electrostatic potential at the ion chelation site is unusually large, and more
than compensates for the substantial energetic penalties associated with partial
dehydration of the ion and displacement of delocalized ions. The very large
predicted binding free energy, approximately -30 kcal/mol, implies that the site
must be occupied for the RNA to fold. These findings agree with previous studies
of the ion-dependent folding of tertiary structure in this RNA, which concluded
that a monovalent ion was bound in a partially dehydrated environment where Mg2+
could not easily compete for binding. By compensating the unfavorable free
energy of buried phosphate groups with a chelated ion, the RNA is able to create
a larger and more complex tertiary fold than would be possible otherwise. (c)
2002 Elsevier Science Ltd.

PMID: 12054794 [PubMed - indexed for MEDLINE]



NR25: J Mol Biol. 2002 May 24;319(1):27-35. 

Initiation factor IF2, thiostrepton and micrococcin prevent the binding of
elongation factor G to the Escherichia coli ribosome.

Cameron DM, Thompson J, March PE, Dahlberg AE.

School of Microbiology and Immunology, University of New South Wales, Sydney,
NSW 2052, Australia.

The bacterial translational GTPases (initiation factor IF2, elongation factors
EF-G and EF-Tu and release factor RF3) are involved in all stages of
translation, and evidence indicates that they bind to overlapping sites on the
ribosome, whereupon GTP hydrolysis is triggered. We provide evidence for a
common ribosomal binding site for EF-G and IF2. IF2 prevents the binding of EF-G
to the ribosome, as shown by Western blot analysis and fusidic acid-stabilized
EF-G.GDP.ribosome complex formation. Additionally, IF2 inhibits EF-G-dependent
GTP hydrolysis on 70 S ribosomes. The antibiotics thiostrepton and micrococcin,
which bind to part of the EF-G binding site and interfere with the function of
the factor, also affect the function of IF2. While thiostrepton is a strong
inhibitor of EF-G-dependent GTP hydrolysis, GTP hydrolysis by IF2 is stimulated
by the drug. Micrococcin stimulates GTP hydrolysis by both factors. We show
directly that these drugs act by destabilizing the interaction of EF-G with the
ribosome, and provide evidence that they have similar effects on IF2.

PMID: 12051934 [PubMed - indexed for MEDLINE]



NR26: J Mol Biol. 2002 May 31;319(2):329-39. 

Limitation of ribosomal protein L11 availability in vivo affects translation
termination.

Van Dyke N, Xu W, Murgola EJ.

Department of Molecular Genetics, Box 11, The University of Texas M. D. Anderson
Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030-4009, USA.

Historically referred to as "the GTPase center", the L11 binding region (L11BR)
of Escherichia coli 23 S rRNA is a highly conserved structure that has been
implicated in several essential functions during protein synthesis. Here, in
vivo expression of an RNA fragment containing that structure was found to affect
translation termination in a codon-specific manner. The cause of these effects
appeared to be titration of ribosomal protein L11, since normal phenotypes could
be restored by simultaneous overproduction of wild-type L11 but not mutant L11.
Subsequently, altered termination phenotypes were produced when the availability
of L11 was limited by overexpression of RNA antisense to L11 mRNA and, finally,
by inactivation of the chromosomal L11 gene, and they too were reversible by
simultaneous expression of cloned L11. Our results indicate that in the intact
cell the L11BR is an integral functional unit important for translation
termination and that the presence of L11 in ribosomes is required for
UAG-dependent termination and is somewhat inhibitory of UGA-dependent
termination. Copyright 2002 Elsevier Science Ltd.

PMID: 12051910 [PubMed - indexed for MEDLINE]



NR27: J Biol Chem. 2002 Aug 9;277(32):28780-6. Epub 2002 Jun 4. 

Rpf2p, an evolutionarily conserved protein, interacts with ribosomal protein L11
and is essential for the processing of 27 SB Pre-rRNA to 25 S rRNA and the 60 S
ribosomal subunit assembly in Saccharomyces cerevisiae.

Morita D, Miyoshi K, Matsui Y, Toh-E A, Shinkawa H, Miyakawa T, Mizuta K.

Department of Molecular Biotechnology, Graduate School of Advanced Sciences of
Matter, Hiroshima University, Kagamiyama 1-4-4, Higashi-Hiroshima 739-8528,
Japan.

Saccharomyces cerevisiae Rrs1p is a nuclear protein that is essential for the
maturation of 25 S rRNA and the 60 S ribosomal subunit assembly. In two-hybrid
screening, using RRS1 as bait, we have cloned YKR081c/RPF2. Rpf2p is essential
for growth and is mainly localized in the nucleolus. The amino acid sequence of
Rpf2p is highly conserved in eukaryotes from yeast to human. Similar to Rrs1p,
Rpf2p shows physical interaction with ribosomal protein L11 and appears to
associate with preribosomal subunits fairly tightly. Northern, methionine
pulse-chase, and sucrose density gradient ultracentrifugation analyses reveal
that the depletion of Rpf2p results in a delayed processing of pre-rRNA, a
decrease of mature 25 S rRNA, and a shortage of 60 S subunits. An analysis of
processing intermediates by primer extension shows that the Rpf2p depletion
leads to an accumulation of 27 SB pre-rRNA, suggesting that Rpf2p is required
for the processing of 27 SB into 25 S rRNA.

PMID: 12048200 [PubMed - indexed for MEDLINE]



NR28: J Biol Chem. 2002 May 24;277(21):18334-9. Epub 2002 Mar 13. 

Normal assembly of 60 S ribosomal subunits is required for the signaling in
response to a secretory defect in Saccharomyces cerevisiae.

Miyoshi K, Tsujii R, Yoshida H, Maki Y, Wada A, Matsui Y, Toh-E A, Mizuta K.

Department of Biological Sciences, Graduate School of Biosphere Sciences,
Graduate School of Advanced Sciences of Matter, Hiroshima University,
Kagamiyama, Higashi-Hiroshima 739-8528, Japan.

A secretory defect leads to transcriptional repression of both ribosomal protein
and rRNA genes in yeast. To elucidate the mechanism of the signaling, we
previously isolated rrs mutants that were unable to respond to a secretory
defect, and we cloned RRS1 encoding a nuclear protein that was required for
ribosome biogenesis (Tsuno, A., Miyoshi, K., Tsujii, R., Miyakawa, T., and
Mizuta, K. (2000) Mol. Cell. Biol. 20, 2066-2074). We identified duplicated
genes encoding ribosomal protein L11, RPL11B as a wild-type allele complementing
the rrs2 mutation, and RPL11A in two-hybrid screening using RRS1 as bait. Rpl11p
was copurified with Rrs1p in immunoprecipitation analysis. Ultracentrifugation
analysis revealed that Rrs1p associated fairly tightly with 60 S preribosomal
subunits. These results suggest that signaling in response to a secretory defect
requires the normal assembly of 60 S ribosomal subunits including Rrs1p and
Rpl11p.

PMID: 11893754 [PubMed - indexed for MEDLINE]



NR29: J Biol Chem. 2002 Feb 8;277(6):3857-62. Epub 2001 Nov 29. 

Translation elongation by a hybrid ribosome in which proteins at the GTPase
center of the Escherichia coli ribosome are replaced with rat counterparts.

Uchiumi T, Honma S, Nomura T, Dabbs ER, Hachimori A.

Institute of High Polymer Research, Faculty of Textile Science and Technology,
Shinshu University, Ueda 386-8567, Japan. uchiumi@giptc.shinshu-u.ac.jp

Ribosomal L10-L7/L12 protein complex and L11 bind to a highly conserved RNA
region around position 1070 in domain II of 23 S rRNA and constitute a part of
the GTPase-associated center in Escherichia coli ribosomes. We replaced these
ribosomal proteins in vitro with the rat counterparts P0-P1/P2 complex and RL12,
and tested them for ribosomal activities. The core 50 S subunit lacking the
proteins on the 1070 RNA domain was prepared under gentle conditions from a
mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50
S subunit through their interactions with the 1070 RNA domain. The resultant
hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed
polyphenylalanine synthesis dependent on the actions of both eukaryotic
elongation factors 1alpha (eEF-1alpha) and 2 (eEF-2) but not of the prokaryotic
equivalent factors EF-Tu and EF-G. The results from replacement of either the
L10-L7/L12 complex or L11 with rat protein showed that the P0-P1/P2 complex, and
not RL12, was responsible for the specificity of the eukaryotic ribosomes to
eukaryotic elongation factors and for the accompanying GTPase activity. The
presence of either E. coli L11 or rat RL12 considerably stimulated the
polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12
proteins play an important role in post-GTPase events of translation elongation.

PMID: 11729183 [PubMed - indexed for MEDLINE]



NR30: J Bacteriol. 2001 Nov;183(22):6532-7. 

Involvement of the N terminus of ribosomal protein L11 in regulation of the RelA
protein of Escherichia coli.

Yang X, Ishiguro EE.

Department of Biochemistry and Microbiology, University of Victoria, British
Columbia, Canada.

Amino acid-deprived rplK (previously known as relC) mutants of Escherichia coli
cannot activate (p)ppGpp synthetase I (RelA) and consequently exhibit relaxed
phenotypes. The rplK gene encodes ribosomal protein L11, suggesting that L11 is
involved in regulating the activity of RelA. To investigate the role of L11 in
the stringent response, a derivative of rplK encoding L11 lacking the N-terminal
36 amino acids (designated 'L11) was constructed. Bacteria overexpressing 'L11
exhibited a relaxed phenotype, and this was associated with an inhibition of
RelA-dependent (p)ppGpp synthesis during amino acid deprivation. In contrast,
bacteria overexpressing normal L11 exhibited a typical stringent response. The
overexpressed 'L11 was incorporated into ribosomes and had no effect on the
ribosome-binding activity of RelA. By several methods (yeast two-hybrid,
affinity blotting, and copurification), no direct interaction was observed
between the C-terminal ribosome-binding domain of RelA and L11. To determine
whether the proline-rich helix of L11 was involved in RelA regulation, the
Pro-22 residue was replaced with Leu by site-directed mutagenesis. The
overexpression of the Leu-22 mutant derivative of L11 resulted in a relaxed
phenotype. These results indicate that the proline-rich helix in the N terminus
of L11 is involved in regulating the activity of RelA.

PMID: 11673421 [PubMed - indexed for MEDLINE]



NR31: Genome Biol. 2001;2(9):RESEARCH 0033. Epub 2001 Aug 30. 

Two C or not two C: recurrent disruption of Zn-ribbons, gene duplication,
lineage-specific gene loss, and horizontal gene transfer in evolution of
bacterial ribosomal proteins.

Makarova KS, Ponomarev VA, Koonin EV.

National Center for Biotechnology Information, National Library of Medicine,
National Institutes of Health, Bethesda, MD 20894, USA. koonin@ncbi.nlm.nih.gov

BACKGROUND: Ribosomal proteins are encoded in all genomes of cellular life forms
and are, generally, well conserved during evolution. In prokaryotes, the genes
for most ribosomal proteins are clustered in several highly conserved operons,
which ensures efficient co-regulation of their expression. Duplications of
ribosomal-protein genes are infrequent, and given their coordinated expression
and functioning, it is generally assumed that ribosomal-protein genes are
unlikely to undergo horizontal transfer. However, with the accumulation of
numerous complete genome sequences of prokaryotes, several paralogous pairs of
ribosomal protein genes have been identified. Here we analyze all such cases and
attempt to reconstruct the evolutionary history of these ribosomal proteins.
RESULTS: Complete bacterial genomes were searched for duplications of ribosomal
proteins. Ribosomal proteins L36, L33, L31, S14 are each duplicated in several
bacterial genomes and ribosomal proteins L11, L28, L7/L12, S1, S15, S18 are so
far duplicated in only one genome each. Sequence analysis of the four ribosomal
proteins, for which paralogs were detected in several genomes, two of the
ribosomal proteins duplicated in one genome (L28 and S18), and the ribosomal
protein L32 showed that each of them comes in two distinct versions. One form
contains a predicted metal-binding Zn-ribbon that consists of four conserved
cysteines (in some cases replaced by histidines), whereas, in the second form,
these metal-chelating residues are completely or partially replaced. Typically,
genomes containing paralogous genes for these ribosomal proteins encode both
versions, designated C+ and C-, respectively. Analysis of phylogenetic trees for
these seven ribosomal proteins, combined with comparison of genomic contexts for
the respective genes, indicates that in most, if not all cases, their evolution
involved a duplication of the ancestral C+ form early in bacterial evolution,
with subsequent alternative loss of the C+ and C- forms in different lineages.
Additionally, evidence was obtained for a role of horizontal gene transfer in
the evolution of these ribosomal proteins, with multiple cases of gene
displacement 'in situ', that is, without a change of the gene order in the
recipient genome. CONCLUSIONS: A more complex picture of evolution of bacterial
ribosomal proteins than previously suspected is emerging from these results,
with major contributions of lineage-specific gene loss and horizontal gene
transfer. The recurrent theme of emergence and disruption of Zn-ribbons in
bacterial ribosomal proteins awaits a functional interpretation.

PMID: 11574053 [PubMed - indexed for MEDLINE]



NR32: J Biol Chem. 2001 Nov 23;276(47):43958-69. Epub 2001 Sep 10. 

The large subunit of the mammalian mitochondrial ribosome. Analysis of the
complement of ribosomal proteins present.

Koc EC, Burkhart W, Blackburn K, Moyer MB, Schlatzer DM, Moseley A, Spremulli
LL.

Department of Chemistry and Campus Box 3290, University of North Carolina,
Chapel Hill, North Carolina 27599-3290, USA.

Identification of all the protein components of the large subunit (39 S) of the
mammalian mitochondrial ribosome has been achieved by carrying out proteolytic
digestions of whole 39 S subunits followed by analysis of the resultant peptides
by liquid chromatography and mass spectrometry. Peptide sequence information was
used to search the human EST data bases and complete coding sequences were
assembled. The human mitochondrial 39 S subunit has 48 distinct proteins. Twenty
eight of these are homologs of the Escherichia coli 50 S ribosomal proteins L1,
L2, L3, L4, L7/L12, L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L20, L21,
L22, L23, L24, L27, L28, L30, L32, L33, L34, L35, and L36. Almost all of these
proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and
Saccharomyces cerevisiae mitochondrial ribosomes. No mitochondrial homologs to
prokaryotic ribosomal proteins L5, L6, L25, L29, and L31 could be found either
in the peptides obtained or by analysis of the available data bases. The
remaining 20 proteins present in the 39 S subunits are specific to mitochondrial
ribosomes. Proteins in this group have no apparent homologs in bacterial,
chloroplast, archaebacterial, or cytosolic ribosomes. All but two of the
proteins has a clear homolog in D. melanogaster while all can be found in the
genome of C. elegans. Ten of the 20 mitochondrial specific 39 S proteins have
homologs in S. cerevisiae. Homologs of 2 of these new classes of ribosomal
proteins could be identified in the Arabidopsis thaliana genome.

PMID: 11551941 [PubMed - indexed for MEDLINE]



NR33: Plant J. 2001 Aug;27(3):179-89. 

Knock-out of the plastid ribosomal protein L11 in Arabidopsis: effects on mRNA
translation and photosynthesis.

Pesaresi P, Varotto C, Meurer J, Jahns P, Salamini F, Leister D.

Zentrum zur Identifikation von Genfunktionen durch Insertionsmutagenese bei
Arabidopsis thaliana (ZIGIA), Max-Planck-Institut fur Zuchtungsforschung,
Carl-von-Linne-Weg 10, 50829 Koln, Germany.

The prpl11-1 mutant of Arabidopsis thaliana was identified among a collection of
T-DNA tagged lines on the basis of a decrease in the effective quantum yield of
photosystem II. The mutation responsible was localized to Prpl11, a single-copy
nuclear gene that encodes PRPL11, a component of the large subunit of the
plastid ribosome. The amino acid sequence of Arabidopsis PRPL11 is very similar
to those of L11 proteins from spinach and prokaryotes. In the prpl11-1 mutant,
photosensitivity and chlorophyll fluorescence parameters are significantly
altered owing to changes in the levels of thylakoid protein complexes and
stromal proteins. The abundance of most plastome transcripts examined, such as
those of genes coding for the photosystem II core complex and RbcL, is not
decreased. Plastid ribosomal RNA accumulates in wild-type amounts, and the
assembly of plastid polysomes on the transcripts of the rbcL, psbA and psbE
genes remains mainly unchanged in mutant plants, indicating that lack of PRPL11
affects neither the abundance of plastid ribosomes nor their assembly into
polysomes. However, in vivo translation assays demonstrate that the rate of
translation of the large subunit of Rubisco (RbcL) is significantly reduced in
prpl11-1 plastids. Our data suggest a major role for PRPL11 in plastid ribosome
activity per se, consistent with its location near the GTPase-binding centre of
the chloroplast 50S ribosomal subunit. Additional effects of the mutation,
including the pale green colour of the leaves and a drastic reduction in growth
rate under greenhouse conditions, are compatible with reduced levels of protein
synthesis in plastids.

PMID: 11532164 [PubMed - indexed for MEDLINE]



NR34: J Biomol NMR. 2001 Jul;20(3):293-4. 

1H, 15N, and 13C assignments and secondary structure identification for
full-length ribosomal protein L11 from Thermus thermophilus.

Markus MA, Triantafillidou D, Choli-Papadopoulou T, Torchia DA.

Publication Types:
    Letter

PMID: 11519754 [PubMed - indexed for MEDLINE]



NR35: J Mol Biol. 2001 Aug 24;311(4):777-87. 

Localization of L11 protein on the ribosome and elucidation of its involvement
in EF-G-dependent translocation.

Agrawal RK, Linde J, Sengupta J, Nierhaus KH, Frank J.

Wadsworth Center, Empire State Plaza, Albany, NY 12201-0509, USA.
agrawal@wadsworth.org

L11 protein is located at the base of the L7/L12 stalk of the 50 S subunit of
the Escherichia coli ribosome. Because of the flexible nature of the region,
recent X-ray crystallographic studies of the 50 S subunit failed to locate the
N-terminal domain of the protein. We have determined the position of the
complete L11 protein by comparing a three-dimensional cryo-EM reconstruction of
the 70 S ribosome, isolated from a mutant lacking ribosomal protein L11, with
the three-dimensional map of the wild-type ribosome. Fitting of the X-ray
coordinates of L11-23 S RNA complex and EF-G into the cryo-EM maps combined with
molecular modeling, reveals that, following EF-G-dependent GTP hydrolysis,
domain V of EF-G intrudes into the cleft between the 23 S ribosomal RNA and the
N-terminal domain of L11 (where the antibiotic thiostrepton binds), causing the
N-terminal domain to move and thereby inducing the formation of the arc-like
connection with the G' domain of EF-G. The results provide a new insight into
the mechanism of EF-G-dependent translocation. Copyright 2001 Academic Press.

PMID: 11518530 [PubMed - indexed for MEDLINE]



NR36: Mol Genet Genomics. 2001 Jun;265(4):569-75. 

Characterization of a wheat cDNA encoding mitochondrial ribosomal protein L11:
qualitative and quantitative tissue-specific differences in its expression.

Handa H, Kobayashi-Uehara A, Murayama S.

Laboratory of Plant Genecology, Hokkaido National Agricultural Experiment
Station, Sapporo, Japan. hirokazu@cryo.affrc.go.jp

We have cloned a cDNA for a ribosomal protein of wheat that is similar to the
bacterial ribosomal protein L11 (RPL11). To determine the subcellular
localization of the gene product, we fused the whole cDNA sequence to the coding
sequence for Green Fluorescent Protein, and expressed the fusion product
transiently in epidermal cells of pea hypocotyls or dayflower leaves. Localized
fluorescence was detectable in mitochondria, indicating that this nuclear cDNA
encodes a mitochondrial ribosomal protein L11 (MRPL11). In lower protists,
mitochondrial RPL11 is encoded by the mitochondrial genome, but higher
organisms, including animals, fungi and plants, do not have genes for RPL11 in
their mitochondrial genomes, suggesting that transfer of the genetic information
for RPL11 from the mitochondrial genome to the nucleus was a very early event in
evolution. Transcripts of this wheat gene (TaMRPL11) for mitochondrial RPL11
were found in all tissues examined, although qualitative and quantitative
differences in expression were noted. The transcript sizes were different in
different plant tissues: 1.0 kb in flowers and roots, and 1.5 kb in shoots. Cold
stress transiently increased the steady-state level of TaMRPL11 mRNA in shoots,
but the transcription of TaMRPL11 was completely inhibited by cold treatment for
longer periods. However, the transcript level in flowers decreased gradually on
exposure to low temperature. On the other hand, the accumulation of TaMRPL11
transcripts in roots was not affected by low temperature. These results suggest
that the expression of MRPL11 in wheat is regulated precisely, in a
tissue-specific manner.

PMID: 11459176 [PubMed - indexed for MEDLINE]



NR37: Mol Microbiol. 2001 May;40(4):909-16. 

Transcription of essential cell division genes is linked to chromosome
replication in Escherichia coli.

Liu G, Begg K, Geddes A, Donachie WD.

Institute of Cell and Molecular Biology, University of Edinburgh, Darwin
Building, King's Buildings, Edinburgh EH9 3JR, UK. Guowen.liu@yale.edu

Cell division normally follows the completion of each round of chromosome
replication in Escherichia coli. Transcription of the essential cell division
genes clustered at the mra region is shown here to depend on continuing
chromosomal DNA replication. After chromosome replication was blocked by either
nalidixic acid treatment or thymine starvation, the transcription of these cell
division genes was repressed significantly. This suggests a way in which cell
division is controlled by chromosome replication.

PMID: 11401698 [PubMed - indexed for MEDLINE]



NR38: J Mol Biol. 2001 May 18;308(5):919-36. 

Quantitative analysis of nucleic acid three-dimensional structures.

Gendron P, Lemieux S, Major F.

Departement d'Informatique et de Recherche Operationnelle, Universite de
Montreal, C.P. 6128, Succ. Centre-Ville, Montreal, Quebec, H3C 3J7, Canada.

A new computer program to annotate DNA and RNA three-dimensional structures,
MC-Annotate, is introduced. The goals of annotation are to efficiently extract
and manipulate structural information, to simplify further structural analyses
and searches, and to objectively represent structural knowledge. The input of
MC-Annotate is a PDB formatted DNA or RNA three-dimensional structure. The
output of MC-Annotate is composed of a structural graph that contains the
annotations, and a series of HTML documents, one for each nucleotide
conformation and base-base interaction present in the input structure. The
atomic coordinates of all nucleotides and the homogeneous transformation
matrices of all base-base interactions are stored in the structural graph.
Symbolic classifications of nucleotide conformations, using sugar puckering
modes and nitrogen base orientations around the glycosyl bond, and base-base
interactions, using stacking and hydrogen bonding information, are introduced.
Peculiarity factors of nucleotide conformations and base-base interactions are
defined to indicate their marginalities with all other examples. The peculiarity
factors allow us to identify irregular regions and possible stereochemical
errors in 3-D structures without interactive visualization. The annotations
attached to each nucleotide conformation include its class, its torsion angles,
a distribution of the root-mean-square deviations with examples of the same
class, the list of examples of the same class, and its peculiarity value. The
annotations attached to each base-base interaction include its class, a
distribution of distances with examples of the same class, the list of examples
of the same class, and its peculiarity value. The distance between two
homogeneous transformation matrices is evaluated using a new metric that
distinguishes between the rotation and the translation of a transformation
matrix in the context of nitrogen bases. MC-Annotate was used to build databases
of nucleotide conformations and base-base interactions. It was applied to the
ribosomal RNA fragment that binds to protein L11, which annotations revealed
peculiar nucleotide conformations and base-base interactions in the regions
where the RNA contacts the protein. The question of whether the current database
of RNA three-dimensional structures is complete is addressed. Copyright 2001
Academic Press.

PMID: 11352582 [PubMed - indexed for MEDLINE]



NR39: Appl Environ Microbiol. 2001 May;67(5):2183-90. 

Organization and transcriptional analysis of a six-gene cluster around the
rplK-rplA operon of Corynebacterium glutamicum encoding the ribosomal proteins
L11 and L1.

Barreiro C, Gonzalez-Lavado E, Martin JF.

Instituto de Biotecnologia (INBIOTEC), Parque Cientifico de Leon, Avda. del
Real, no. 1, 24006 Leon, Spain.

A cluster of six genes, tRNA(Trp)-secE-nusG-rplK-rplA-pkwR, was cloned and
sequenced from a Corynebacterium glutamicum cosmid library and shown to be
contiguous in the C. glutamicum genome. These genes encode a tryptophanyl tRNA,
the protein translocase component SecE, the antiterminator protein NusG, and the
ribosomal proteins L11 and L1 in addition to PkwR, a putative regulatory protein
of the LacI-GalR family. S1 nuclease mapping analysis revealed that nusG and
rplK are expressed as separate transcriptional units and rplK and rplA are
cotranscribed as a single mRNA. A 19-nucleotide inverted repeat that appears to
correspond to a transcriptional terminator was located in the 3' region
downstream from nusG. Northern analysis with different probes confirmed the S1
mapping results and showed that the secE-rplA four-gene region gives rise to
four transcripts. secE was transcribed as a 0.5-kb monocistronic mRNA, nusG
formed two transcripts of 1.4 and 1.0 kb from different initiation sites, and
the two ribosomal protein genes rplK and rplA were cotranscribed as a single
mRNA of 1.6 kb. A consensus L1 protein binding sequence was identified in the
leader region of the rplK-rplA transcript, suggesting that expression of the
rplK-rplA cluster was regulated by autogenous regulation exerted by the L1
protein at the translation level. The promoters of the nusG and rplK-rplA genes
were subcloned in a novel corynebacterial promoter-probe vector and shown to
confer strong expression of the reporter gene.

PMID: 11319098 [PubMed - indexed for MEDLINE]



NR40: J Biol Chem. 2001 Apr 27;276(17):14117-23. Epub 2001 Jan 30. 

Cryo-electron microscopic localization of protein L7/L12 within the Escherichia
coli 70 S ribosome by difference mapping and Nanogold labeling.

Montesano-Roditis L, Glitz DG, Traut RR, Stewart PL.

Department of Biological Chemistry, University of California School of Medicine,
Los Angeles, California 90095-1737, USA.

The Escherichia coli ribosomal protein L7/L12 is central to the translocation
step of translation, and it is known to be flexible under some conditions. The
assignment of electron density to L7/L12 was not possible in the recent 2.4 A
resolution x-ray crystallographic structure (Ban, N., Nissen, P., Hansen, J.,
Moore, P. B., and Steitz, T. A. (2000) Science 289, 905-920). We have localized
the two dimers of L7/L12 within the structure of the 70 S ribosome using two
reconstitution approaches together with cryo-electron microscopy and single
particle reconstruction. First, the structures were determined for ribosomal
cores from which protein L7/L12 had been removed by treatment with NH(4)Cl and
ethanol and for reconstituted ribosomes in which purified L7/L12 had been
restored to core particles. Difference mapping revealed that the reconstituted
ribosomes had additional density within the L7/L12 shoulder next to protein L11.
Second, ribosomes were reconstituted using an L7/L12 variant in which a single
cysteine at position 89 in the C-terminal domain was modified with Nanogold
(Nanoprobes, Inc.), a 14 A gold derivative. The reconstruction from
cryo-electron microscopy images and difference mapping placed the gold at four
interfacial positions. The finding of multiple sites for the C-terminal domain
of L7/L12 suggests that the conformation of this protein may change during the
steps of elongation and translocation.

PMID: 11278411 [PubMed - indexed for MEDLINE]



NR41: Nat Struct Biol. 2001 Apr;8(4):339-43. 

A universal mode of helix packing in RNA.

Doherty EA, Batey RT, Masquida B, Doudna JA.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven,
Connecticut 06520, USA.

RNA molecules fold into specific three-dimensional shapes to perform structural
and catalytic functions. Large RNAs can form compact globular structures, but
the chemical basis for close helical packing within these molecules has been
unclear. Analysis of transfer, catalysis, in vitro-selected and ribosomal RNAs
reveal that helical packing predominantly involves the interaction of
single-stranded adenosines with a helix minor groove. Using the Tetrahymena
thermophila group I ribozyme, we show here that the near-perfect shape
complementarity between the adenine base and the minor groove allows for optimal
van der Waals contacts, extensive hydrogen bonding and hydrophobic surface
burial, creating a highly energetically favorable interaction. Adenosine is
recognized in a chemically similar fashion by a combination of protein and RNA
components in the ribonucleoprotein core of the signal recognition particle.
These results provide a thermodynamic explanation for the noted abundance of
conserved adenosines within the unpaired regions of RNA secondary structures.

PMID: 11276255 [PubMed - indexed for MEDLINE]



PR42: Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):3992-7. 

The immunoprotective MHC II epitope of a chemically induced tumor harbors a
unique mutation in a ribosomal protein.

Matsutake T, Srivastava PK.

Center for Immunotherapy of Cancer and Infectious Diseases, University of
Connecticut School of Medicine, Farmington, CT 06030-1601, USA.

CD4(+) T lymphocyte clones, generated from mice immunized with the
methylcholanthrene-induced fibrosarcoma Meth A (H-2(d)), are restricted by
I-E(d) and recognize a unique antigen on Meth A. The antigen has been purified
and characterized as the ribosomal protein L11. The antigenic epitope is
contained within the sequence EYELRKHNFSDTG and is generated by substitution of
Asn by His (italic) caused by a single point mutation. The tumor contains the
wild-type and the mutated alleles. Immunization of BALB/cJ mice with the mutated
epitope but not with the wild-type epitope protects mice against a subsequent
challenge with the Meth A sarcoma. Adoptive transfer of CD4(+) clones into
BALB/c mice renders the mice specifically resistant to Meth A sarcoma. The
mutated L11 epitope is thus shown to be an immunoprotective epitope in vivo by
several criteria.

PMID: 11274422 [PubMed - indexed for MEDLINE]



NR43: J Bacteriol. 2001 Apr;183(7):2316-21. 

Loss of ribosomal protein L11 blocks stress activation of the Bacillus subtilis
transcription factor sigma(B).

Zhang S, Scott JM, Haldenwang WG.

Department of Microbiology, MC 7758, University of Texas Health Science Center,
San Antonio, Texas 78229-3900, USA.

sigma(B), the general stress response sigma factor of Bacillus subtilis, is
activated when the cell's energy levels decline or the bacterium is exposed to
environmental stress (e.g., heat shock, ethanol). Physical stress activates
sigma(B) through a collection of regulatory kinases and phosphatases (the Rsb
proteins) which catalyze the release of sigma(B) from an anti-sigma(B) factor
inhibitor. The means by which diverse stresses communicate with the Rsb proteins
is unknown; however, a role for the ribosome in this process was suggested when
several of the upstream members of the sigma(B) stress activation cascade (RsbR,
-S, and -T) were found to cofractionate with ribosomes in crude B. subtilis
extracts. We now present evidence for the involvement of a ribosome-mediated
process in the stress activation of sigma(B). B. subtilis strains resistant to
the antibiotic thiostrepton, due to the loss of ribosomal protein L11 (RplK),
were found to be blocked in the stress activation of sigma(B). Neither the
energy-responsive activation of sigma(B) nor stress-dependent chaperone gene
induction (a sigma(B)-independent stress response) was inhibited by the loss of
L11. The Rsb proteins required for stress activation of sigma(B) are shown to be
active in the RplK(-) strain but fail to be triggered by stress. The data
demonstrate that the B. subtilis ribosomes provide an essential input for the
stress activation of sigma(B) and suggest that the ribosomes may themselves be
the sensors for stress in this system.

PMID: 11244072 [PubMed - indexed for MEDLINE]


NR44: Microbiology. 2001 Mar;147(Pt 3):691-700. 

A Corynebacterium glutamicum mutant with a defined deletion within the rplK gene
is impaired in (p)ppGpp accumulation upon amino acid starvation.

Wehmeier L, Brockmann-Gretza O, Pisabarro A, Tauch A, Puhler A, Martin JF,
Kalinowski J.

Lehrstuhl fur Genetik, Fakultat fur Biologie, Universitat Bielefeld, D-33501
Bielefeld, Germany.

The rplK gene of Corynebacterium glutamicum ATCC13032 comprises 438 nucleotides
and encodes a protein of 145 amino acids with a molecular mass of 15.3 kDa. The
amino acid sequence revealed extensive similarities to the large ribosomal
subunit protein L11 from several Gram-positive and Gram-negative bacteria. The
C. glutamicum rplK gene is located downstream of secE, representing part of the
protein export apparatus, and of nusG, encoding a transcription antiterminator
protein. The rplK gene is followed by an ORF homologous to rplA encoding the 50S
ribosomal protein L1. Northern analysis revealed that transcription of the
rplK-rplA cluster resulted in two different transcripts of 1.5 and 0.6 kb. The
1.5 kb transcript corresponds to the entire rplK-rplA cluster and the short
transcript originates from the rplK gene. A C. glutamicum rplK mutant strain
carrying a 12 bp in-frame deletion within rplK, which resulted in the loss of
the tetrapeptide Pro-Ala-Leu-Gly in the L11 protein, was constructed. The mutant
failed to accumulate (p)ppGpp in response to amino acid starvation and exhibited
an increased tolerance to the antibiotic thiostrepton. Evidently, the C.
glutamicum rplK gene is required for (p)ppGpp accumulation upon nutritional
starvation.

PMID: 11238976 [PubMed - indexed for MEDLINE]



NR45: Biol Chem. 2000 Nov;381(11):1079-87. 

Identification of the 50S ribosomal proteins from the Eubacterium Thermus
thermophilus.

Katsani KR, Tsiboli P, Anagnostopoulos K, Urlaub H, Choli-Papadopoulou T.

Laboratory of Biochemistry, School of Chemistry, Aristotle University of
Thessaloniki, Greece.

The total protein mixture from the 50S subunit (TP-50) of the eubacterium
Thermus thermophilus was characterized after blotting onto PVDF membranes from
two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and sequencing. The
proteins were numbered according to their primary structure similarity with
their counterparts from other species. One of them has been marked with an
asterisk, namely L*23, because unlike the other known ribosomal proteins it
shows a very low degree of homology. A highly acidic 5S rRNA binding protein,
TL5, was characterized and compared with the available primary structure
information. Proteins L1 and L4 migrate similarly on 2D-PAGE. Protein L4,
essential for protein biosynthesis, is N-terminally blocked and shows a
strikingly low homology to other L4 proteins. In addition to L4, two other
proteins, namely L10 and L11, were found to be N-terminally blocked. In
conclusion, 33 proteins from the large subunit were identified, including TL5.
Homologs to rpL25 and rpL26 were not found.

PMID: 11154066 [PubMed - indexed for MEDLINE]



NR46: Differentiation. 2000 Oct;66(2-3):81-92. 

Down regulation of ribosomal protein mRNAs during neuronal differentiation of
human NTERA2 cells.

Bevort M, Leffers H.

Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark.

We have analysed the expression of 32 ribosomal protein (RP) mRNAs during
retinoic acid induced neuronal differentiation of human NTERA2 cells. Except for
a new S27 variant (S27v), all were down regulated both in selectively replated
differentiated neurons and the most differentiated continuous cultures, i.e.,
non-replated cultures. However, the expression profiles of the individual RP
mRNAs were different, most (L3, L7, L8, L10, L13, L23a, L27a, L36a, L39, P0, S2,
S3, S3a, S4X, S6, S9, S12, S13, S16, S19, S20, S23, and S27a) exhibited a
constant down regulation, whereas a few were either initially constant (L11,
L32, S8, and S11) or up regulated (L6, L15, L17, L31, and S27y) and then down
regulated. The expression of S27v remained elevated in the most differentiated
continuous cultures but was down regulated in replated differentiated neurons.
The down regulation of RP mRNAs was variable: the expression levels in
differentiated replated neurons were between 10% (S3) and 90% (S11) of the
levels in undifferentiated cells. The ratio between rRNA and RP mRNA changed
during the differentiation; in differentiated neurons there were, on average,
about half the number of RP mRNAs per rRNA as compared to undifferentiated
cells. The expression profiles of a few translation-related proteins were also
determined. EF1alpha1, EF1beta1, and EF1delta were down regulated, whereas the
expression of the neuron and muscle specific EF1alpha2 increased. The reduction
in the expression of RP mRNAs was coordinated with a reduction in the expression
level of the proliferation marker PCNA. The expression levels of most RP mRNAs
were lower in purified differentiated post-mitotic neurons than in the most
differentiated continuous cultures, despite similar levels of PCNA, suggesting
that both the differentiation state and the proliferative status of the cells
affect the expression of RP mRNAs.

PMID: 11100899 [PubMed - indexed for MEDLINE]



NR47: Nucleic Acids Res. 2000 Nov 15;28(22):4497-505. 

Conformational changes induced in the Saccharomyces cerevisiae GTPase-associated
rRNA by ribosomal stalk components and a translocation inhibitor.

Briones C, Ballesta JP.

Centro de Biologia Molecular 'Severo Ochoa', Consejo Superior de Investigaciones
Cientificas y Universidad Autonoma de Madrid, Canto Blanco, 28049 Madrid, Spain.

The yeast ribosomal GTPase associated center is made of parts of the 26S rRNA
domains II and VI, and a number of proteins including P0, P1alpha, P1beta,
P2alpha, P2beta and L12. Mapping of the rRNA neighborhood of the proteins was
performed by footprinting in ribosomes from yeast strains lacking different
GTPase components. The absence of protein P0 dramatically increases the
sensitivity of the defective ribosome to degradation hampering the RNA
footprinting. In ribosomes lacking the P1/P2 complex, protection of a number of
nucleotides is detected around positions 840, 880, 1100, 1220-1280 and 1350 in
domain II as well as in several positions in the domain VI alpha-sarcin region.
The protection pattern resembles the one reported for the interaction of
elongation factors in bacterial systems. The results exclude a direct
interaction of these proteins with the rRNA and are compatible with an increase
in the ribosome affinity for EF-2 in the absence of the acidic P proteins.
Interestingly, a sordarin derivative inhibitor of EF-2 causes an opposite
effect, increasing the reactivity in positions protected by the absence of
P1/P2. Similarly, a deficiency in protein L12 exposes nucleotides G1235, G1242,
A1262, A1269, A1270 and A1272 to chemical modification, thus situating the
protein binding site in the most conserved part of the 26S rRNA, equivalent to
the bacterial protein L11 binding site.

PMID: 11071938 [PubMed - indexed for MEDLINE]



NR48: Mol Biol Cell. 2000 Nov;11(11):3777-89. 

Factors affecting nuclear export of the 60S ribosomal subunit in vivo.

Stage-Zimmermann T, Schmidt U, Silver PA.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.

In Saccharomyces cerevisiae, the 60S ribosomal subunit assembles in the
nucleolus and then is exported to the cytoplasm, where it joins the 40S subunit
for translation. Export of the 60S subunit from the nucleus is known to be an
energy-dependent and factor-mediated process, but very little is known about the
specifics of its transport. To begin to address this problem, an assay was
developed to follow the localization of the 60S ribosomal subunit in S.
cerevisiae. Ribosomal protein L11b (Rpl11b), one of the approximately 45
ribosomal proteins of the 60S subunit, was tagged at its carboxyl terminus with
the green fluorescent protein (GFP) to enable visualization of the 60S subunit
in living cells. A panel of mutant yeast strains was screened for their
accumulation of Rpl11b-GFP in the nucleus as an indicator of their involvement
in ribosome synthesis and/or transport. This panel included conditional alleles
of several rRNA-processing factors, nucleoporins, general transport factors, and
karyopherins. As predicted, conditional alleles of rRNA-processing factors that
affect 60S ribosomal subunit assembly accumulated Rpl11b-GFP in the nucleus. In
addition, several of the nucleoporin mutants as well as a few of the karyopherin
and transport factor mutants also mislocalized Rpl11b-GFP. In particular,
deletion of the previously uncharacterized karyopherin KAP120 caused
accumulation of Rpl11b-GFP in the nucleus, whereas ribosomal protein import was
not impaired. Together, these data further define the requirements for ribosomal
subunit export and suggest a biological function for KAP120.

PMID: 11071906 [PubMed - indexed for MEDLINE]



NR49: Acta Microbiol Pol. 2000;49(1):19-29. 

Sequence, structural, and evolutionary analysis of prokaryotic ribosomal protein
L11 methyltransferases.

Bujnicki JM.

Henry Ford Health System, Molecular Biology Research Program, Detroit, MI 48202,
USA. iamb@bioinfo.pl

The Escherichia coli PrmA enzyme catalyzes methylation of the large ribosomal
subunit protein L11. Database homology searches, multiple sequence alignment,
and structure prediction allowed to dissect the primary structure of PrmA into
two domains and assign putative functional or structural roles to invariant or
highly conserved residues. Evolutionary relationships within the PrmA family
were also analyzed. The topology of the branching order agrees to a large extent
with the consensus phylogeny of Eubacteria, with the exception of beta and
epsilon subdivisions of Proteobacteria, which most probably had their original
prmA genes replaced by copies acquired via the lateral gene transfer from
gamma-Proteobacteria and some close relative of the ancestor of gramnegative
bacteria, respectively.

PMID: 10997488 [PubMed - indexed for MEDLINE]



NR50: J Biol Chem. 2000 Nov 10;275(45):35116-21. 

A covariant change of the two highly conserved bases in the GTPase-associated
center of 28 S rRNA in silkworms and other moths.

Uchiumi T, Nomura T, Shimizu T, Katakai Y, Mita K, Koike Y, Nakagaki M, Taira H,
Hachimori A.

Institute of High Polymer Research and the Department of Applied Biological
Science, Faculty of Textile Science and Technology, Shinshu University, Ueda
386-8567, Japan. uchimi@giptc.shinshu-u.ac.jp

The GTPase-associated center in 23/28 S rRNA is one of the most conserved
functional domains throughout all organisms. We detected a unique sequence of
this domain in Bombyx mori species in which the bases at positions 1094 and 1098
(numbering from Escherichia coli 23 S rRNA) are C and G instead of the otherwise
universally conserved bases U and A, respectively. These changes were also
observed in four other species of moths, but not in organisms other than the
moths. Characteristics of the B. mori rRNA domain were investigated by native
polyacrylamide gel electrophoresis using RNA fragments containing residues
1030-1128. Although two bands of protein-free RNA appeared on gel, they shifted
to a single band when bound to Bombyx ribosomal proteins Bm-L12 and Bm-P
complex, equivalent to E. coli L11 and L8, respectively. Bombyx RNA showed lower
binding capacity than rat RNA for the ribosomal proteins and anti-28 S
autoantibody, specific for a folded structure of the eukaryotic
GTPase-associated domain. When the C(1094)/G(1098) bases in Bombyx RNA were
replaced by the conserved U/A bases, the protein-free RNA migrated as a single
band, and the complex formation with Bm-L12, Bm-P complex, and anti-28 S
autoantibody was comparable to that of rat RNA. The results suggest that the
GTPase-associated domain of moth-type insects has a labile structural feature
that is caused by an unusual covariant change of the U(1094)/A(1098) bases to
C/G.

PMID: 10960474 [PubMed - indexed for MEDLINE]



NR51: Methods Enzymol. 2000;318:251-68. 

RNA-protein interactions in ribosomes: in vitro selection from randomly
fragmented rRNA.

Stelzl U, Spahn CM, Nierhaus KH.

Max-Planck-Institut fur Molekulare Genetik, Berlin, Germany.

PMID: 10889993 [PubMed - indexed for MEDLINE]



NR52: J Biol Chem. 2000 Sep 15;275(37):28466-82. 

The plastid ribosomal proteins. Identification of all the proteins in the 50 S
subunit of an organelle ribosome (chloroplast).

Yamaguchi K, Subramanian AR.

Department of Biochemistry, The University of Arizona, Tucson, Arizona 85712,
USA.

We have completed identification of all the ribosomal proteins (RPs) in spinach
plastid (chloroplast) ribosomal 50 S subunit via a proteomic approach using
two-dimensional electrophoresis, electroblotting/protein sequencing, high
performance liquid chromatography purification, polymerase chain reaction-based
screening of cDNA library/nucleotide sequencing, and mass spectrometry
(reversed-phase HPLC coupled to electrospray ionization mass spectrometry and
electrospray ionization mass spectrometry). Spinach plastid 50 S subunit
comprises 33 proteins, of which 31 are orthologues of Escherichia coli RPs and
two are plastid-specific RPs (PSRP-5 and PSRP-6) having no homologues in other
types of ribosomes. Orthologues of E. coli L25 and L30 are absent in spinach
plastid ribosome. 25 of the plastid 50 S RPs are encoded in the nuclear genome
and synthesized on cytosolic ribosomes, whereas eight of the plastid RPs are
encoded in the plastid organelle genome and synthesized on plastid ribosomes.
Sites for transit peptide cleavages in the cytosolic RP precursors and formyl
Met processing in the plastid-synthesized RPs were established.
Post-translational modifications were observed in several mature plastid RPs,
including multiple forms of L10, L18, L31, and PSRP-5 and N-terminal/internal
modifications in L2, L11 and L16. Comparison of the RPs in gradient-purified 70
S ribosome with those in the 30 and 50 S subunits revealed an additional
protein, in approximately stoichiometric amount, specific to the 70 S ribosome.
It was identified to be plastid ribosome recycling factor. Combining with our
recent study of the proteins in plastid 30 S subunit (Yamaguchi, K., von
Knoblauch, K., and Subramanian, A. R. (2000) J. Biol. Chem. 275, 28455-28465),
we show that spinach plastid ribosome comprises 59 proteins (33 in 50 S subunit
and 25 in 30 S subunit and ribosome recycling factor in 70 S), of which 53 are
E. coli orthologues and 6 are plastid-specific proteins (PSRP-1 to PSRP-6). We
propose the hypothesis that PSRPs were evolved to perform functions unique to
plastid translation and its regulation, including protein
targeting/translocation to thylakoid membrane via plastid 50 S subunit.

PMID: 10874046 [PubMed - indexed for MEDLINE]



NR53: Structure Fold Des. 2000 May 15;8(5):527-40. 

Crystal structure of the ffh and EF-G binding sites in the conserved domain IV
of Escherichia coli 4.5S RNA.

Jovine L, Hainzl T, Oubridge C, Scott WG, Li J, Sixma TK, Wonacott A, Skarzynski
T, Nagai K.

MRC Laboratory of Molecular Biology, Cambridge, England. jovinl02@doc.mssm.edu

BACKGROUND: Bacterial signal recognition particle (SRP), consisting of 4.5S RNA
and Ffh protein, plays an essential role in targeting signal-peptide-containing
proteins to the secretory apparatus in the cell membrane. The 4.5S RNA increases
the affinity of Ffh for signal peptides and is essential for the interaction
between SRP and its receptor, protein FtsY. The 4.5S RNA also interacts with
elongation factor G (EF-G) in the ribosome and this interaction is required for
efficient translation. RESULTS: We have determined by multiple anomalous
dispersion (MAD) with Lu(3+) the 2.7 A crystal structure of a 4.5S RNA fragment
containing binding sites for both Ffh and EF-G. This fragment consists of three
helices connected by a symmetric and an asymmetric internal loop. In contrast to
NMR-derived structures reported previously, the symmetric loop is entirely
constituted by non-canonical base pairs. These pairs continuously stack and
project unusual sets of hydrogen-bond donors and acceptors into the shallow
minor groove. The structure can therefore be regarded as two double helical rods
hinged by the asymmetric loop that protrudes from one strand. CONCLUSIONS: Based
on our crystal structure and results of chemical protection experiments reported
previously, we predicted that Ffh binds to the minor groove of the symmetric
loop. An identical decanucleotide sequence is found in the EF-G binding sites of
both 4.5S RNA and 23S rRNA. The decanucleotide structure in the 4.5S RNA and the
ribosomal protein L11-RNA complex crystals suggests how 4.5S RNA and 23S rRNA
might interact with EF-G and function in translating ribosomes.

PMID: 10801497 [PubMed - indexed for MEDLINE]



NR54: J Mol Biol. 2000 Apr 21;298(1):35-59. 

The 3D arrangement of the 23 S and 5 S rRNA in the Escherichia coli 50 S
ribosomal subunit based on a cryo-electron microscopic reconstruction at 7.5 A
resolution.

Mueller F, Sommer I, Baranov P, Matadeen R, Stoldt M, Wohnert J, Gorlach M, van
Heel M, Brimacombe R.

Max-Planck-Institut fur Molekulare Genetik, Ihnestrasse 73, Berlin, 14195,
Germany.

The Escherichia coli 23 S and 5 S rRNA molecules have been fitted helix by helix
to a cryo-electron microscopic (EM) reconstruction of the 50 S ribosomal
subunit, using an unfiltered version of the recently published 50 S
reconstruction at 7.5 A resolution. At this resolution, the EM density shows a
well-defined network of fine structural elements, in which the major and minor
grooves of the rRNA helices can be discerned at many locations. The 3D folding
of the rRNA molecules within this EM density is constrained by their
well-established secondary structures, and further constraints are provided by
intra and inter-rRNA crosslinking data, as well as by tertiary interactions and
pseudoknots. RNA-protein cross-link and foot-print sites on the 23 S and 5 S
rRNA were used to position the rRNA elements concerned in relation to the known
arrangement of the ribosomal proteins as determined by immuno-electron
microscopy. The published X-ray or NMR structures of seven 50 S ribosomal
proteins or RNA-protein complexes were incorporated into the EM density. The 3D
locations of cross-link and foot-print sites to the 23 S rRNA from tRNA bound to
the ribosomal A, P or E sites were correlated with the positions of the tRNA
molecules directly observed in earlier reconstructions of the 70 S ribosome at
13 A or 20 A. Similarly, the positions of cross-link sites within the peptidyl
transferase ring of the 23 S rRNA from the aminoacyl residue of tRNA were
correlated with the locations of the CCA ends of the A and P site tRNA. Sites on
the 23 S rRNA that are cross-linked to the N termini of peptides of different
lengths were all found to lie within or close to the internal tunnel connecting
the peptidyl transferase region with the presumed peptide exit site on the
solvent side of the 50 S subunit. The post-transcriptionally modified bases in
the 23 S rRNA form a cluster close to the peptidyl transferase area. The minimum
conserved core elements of the secondary structure of the 23 S rRNA form a
compact block within the 3D structure and, conversely, the points corresponding
to the locations of expansion segments in 28 S rRNA all lie on the outside of
the structure. Copyright 2000 Academic Press.

PMID: 10756104 [PubMed - indexed for MEDLINE]



NR55: Nucleic Acids Res. 2000 Apr 15;28(8):1778-84. 

The RNA-binding domain of ribosomal protein L11 recognizes an rRNA tertiary
structure stabilized by both thiostrepton and magnesium ion.

Blyn LB, Risen LM, Griffey RH, Draper DE.

Ibis Therapeutics, a Division of Isis Pharmaceuticals, 2292 Faraday Avenue,
Carlsbad, CA 92008, USA. lblyn@isisph.com

Antibiotics that inhibit ribosomal function may do so by one of several
mechanisms, including the induction of incorrect RNA folding or prevention of
protein and/or RNA conformational transitions. Thiostrepton, which binds to the
'GTPase center' of the large subunit, has been postulated to prevent
conformational changes in either the L11 protein or rRNA to which it binds.
Scintillation proximity assays designed to look at the binding of the L11
C-terminal RNA-binding domain to a 23S ribosomal RNA (rRNA) fragment, as well as
the ability of thiostrepton to induce that binding, were used to demonstrate the
role of Mg(2+), L11 and thio-strepton in the formation and maintenance of the
rRNA fragment tertiary structure. Experiments using these assays with both an
Escherichia coli rRNA fragment and a thermostable variant of that RNA show that
Mg(2+), L11 and thiostrepton all induce the RNA to fold to an essentially
identical tertiary structure.

PMID: 10734197 [PubMed - indexed for MEDLINE]



NR56: J Mol Biol. 2000 Jan 21;295(3):569-80. 

Contributions of basic residues to ribosomal protein L11 recognition of RNA.

GuhaThakurta D, Draper DE.

Department of Chemistry, Johns Hopkins University, Baltimore, MD, 21218, USA.

The C-terminal domain of ribosomal protein L11, L11-C76, binds in the distorted
minor groove of a helix within a 58 nucleotide domain of 23 S rRNA. To study the
electrostatic component of RNA recognition in this protein, arginine and lysine
residues have been individually mutated to alanine or methionine residues at the
nine sequence positions that are conserved as basic residues among bacterial L11
homologs. In measurements of the salt dependence of RNA-binding, five of these
mutants have a reduced value of - partial differentiallog(K(obs))/ partial
differentiallog[KCl] as compared to the parent L11-C76 sequence, indicating that
these residues interact with the RNA electrostatic field. These five residues
are located at the perimeter of the RNA-binding surface of the protein; all five
of them form salt bridges with phosphates in the crystal structure of the
complex. A sixth residue, Lys47, was found to make an electrostatic contribution
to binding when measurements were made at pH 6.0, but not at pH 7.0; its pK in
the free protein must be <6.5. The unusual behavior of Lys47 is explained by its
burial in the hydrophobic core of the free protein, and unburial in the
RNA-bound protein, where it forms a salt bridge with a phosphate. The
contributions of these six residues to the electrostatic component of binding
are not additive; thus the magnitude of the salt dependence cannot be used to
count the number of ionic interactions in this complex. By interacting with
irregular features of the RNA backbone, including an S-turn, these basic
residues contribute to the specificity of L11 for its target site. Copyright
2000 Academic Press.

PMID: 10623547 [PubMed - indexed for MEDLINE]



NR57: Differentiation. 1999 Oct;65(2):73-88. 

Ribosomal protein gene expression is cell type specific during development in
Dictyostelium discoideum.

Agarwal AK, Parrish SN, Blumberg DD.

Department of Biological Science, University of Maryland Baltimore County, 1000
Hilltop Circle, Baltimore, MD 21250, USA.

Starvation for amino acids initiates the developmental cycle in the cellular
slime mold, Dictyostelium discoideum. Upon starvation one of the earliest
developmental events is the selective loss of the ribosomal protein mRNAs from
polysomes. This loss depends upon sequences in the 5' non-translated leader of
the ribosomal protein (r-protein) mRNAs. Here evidence is presented which
indicates that those cells which will become prestalk cells express the
ribosomal protein genes during development under starvation conditions. Cells
which enter the prespore pathway shut off r-protein synthesis. The promoter and
5' non-translated leader sequences from two ribosomal protein genes, the rp-L11
and the rp-S9 genes, are fused to the Escherichia coli beta-galactosidase
reporter gene. While beta-galactosidase enzyme activity is detected in situ in
most growing cells, by 15 h of development beta-galactosidase enzyme activity is
largely lost from the prespore cells although strong beta-galactosidase enzyme
activity is present in the prestalk cells. These observations suggest the
possibility that the ribosomal protein mRNAs are excluded from polysomes in a
cell-type-specific manner.

PMID: 10550541 [PubMed - indexed for MEDLINE]



NR58: Biofizika. 1999 Jul-Aug;44(4):601-10. 

[Computer analysis of regulatory signals in complete bacterial genomes.
Translation initiation of ribosomal protein operons]

[Article in Russian]

Vitreshchak A, Bansal AK, Titov II, Gel'fand MS.

Institute of Problems of Data Transmission, Russian Academy of Sciences, Moscow,
Russia.

Signals of translation initiation of operons of Haemophilus influenzae ribosomal
proteins were predicted. This process is regulated by the formation of secondary
RNA structures to which one of the proteins encoded in a particular operon
binds. In some cases, these structures imitate the region of protein binding to
rRNA. Predictions are made by comparing with homologous operons of Escherichia
coli and analogous regions of rRNA and by estimating the energy of secondary
structure formation. It is shown that this regulatory mechanism occurs: in
operons L11, S10, S15, spc, and alpha of H.influenzae and, probably, in operon
S15 of Helicobacter pylori, Bacillus subtilis, and Mycoplasma genitalium.

PMID: 10544808 [PubMed - indexed for MEDLINE]



NR59: Microb Comp Genomics. 1999;4(1):47-58. 

Molecular cloning of the dnaK gene region from Bacillus sphaericus in the
context of genomic comparisons.

Ahmad S, Selvapandiyan A, Gasbarri M, Bhatnagar RK.

International Centre for Genetic Engineering and Biotechnology, New Delhi,
India.

The dnaK gene region of Bacillus sphaericus was cloned as a 3.8 kb HindIII
fragment and an overlapping 1.7 kb EcoRI fragment by using an internal B.
sphaericus specific dnaK gene probe generated by polymerase chain reaction
(PCR). Complete DNA sequencing of the two fragments revealed three complete open
reading frames (ORFs). These ORFs exhibited a high degree of identity to the
grpE dnaK, and dnaJ heat shock genes from other gram-positive bacteria. The
order of the genes was found to be grpE-dnaK-dnaJ. Additionally, the 5'-end and
3'-end contained amino acid sequences that were homologous to the C-terminal
sequence of the hrcA gene and the N-terminal sequence of ORF35 (yqeT),
respectively, from Bacillus subtilis. The entire hrcA gene from B. sphaericus
was then isolated by high-fidelity PCR and completely sequenced. A transcription
stop site is located between the dnaK and dnaJ genes but not after the dnaJ
gene. Consistent with this observation, the dnaJ gene is immediately followed by
an ORF that shows a high degree of identity to ORF35 from B. subtilis,
Staphylococcus aureus, and Clostridium acetobutylicum. The presence of ORF35 is
not indicated in other genera representing the gram-positive bacteria. The amino
acid sequence of ORF35 exhibited nearly 30% identity with the methyltransferase
for large subunit ribosomal protein L11 from gram-negative Proteobacteria and
the related protein from cyanobacteria, other gram-negative bacteria, and
Archaea, suggesting the presence of the gene for this protein in the common
ancestor of Bacteria and Archaea. The absence of the ORF35 gene in Mycobacterium
tuberculosis and other gram-positive bacteria indicates that the loss of this
gene must have occurred in an ancestor of other gram-positive bacteria following
their divergence from the ancestor of Bacillus/Clostridium/staphylococcus
lineage.

PMID: 10518301 [PubMed - indexed for MEDLINE]



NR60: Nature. 1999 Aug 26;400(6747):841-7. 

Comment in:
    Nature. 1999 Aug 26;400(6747):811-2.

Placement of protein and RNA structures into a 5 A-resolution map of the 50S
ribosomal subunit.

Ban N, Nissen P, Hansen J, Capel M, Moore PB, Steitz TA.

Department of Molecular Biophysics & Biochemistry, Yale University, Howard
Hughes Medical Institute, New Haven, Connecticut 06520-8114, USA.

We have calculated at 5.0 A resolution an electron-density map of the large 50S
ribosomal subunit from the bacterium Haloarcula marismortui by using phases
derived from four heavy-atom derivatives, intercrystal density averaging and
density-modification procedures. More than 300 base pairs of A-form RNA duplex
have been fitted into this map, as have regions of non-A-form duplex,
single-stranded segments and tetraloops. The long rods of RNA crisscrossing the
subunit arise from the stacking of short, separate double helices, not all of
which are A-form, and in many places proteins crosslink two or more of these
rods. The polypeptide exit channel was marked by tungsten cluster compounds
bound in one heavy-atom-derivatized crystal. We have determined the structure of
the translation-factor-binding centre by fitting the crystal structures of the
ribosomal proteins L6, L11 and L14, the sarcin-ricin loop RNA, and the RNA
sequence that binds L11 into the electron density. We can position either
elongation factor G or elongation factor Tu complexed with an aminoacylated
transfer RNA and GTP onto the factor-binding centre in a manner that is
consistent with results from biochemical and electron microscopy studies.

PMID: 10476961 [PubMed - indexed for MEDLINE]



NR61: Genetics. 1999 Aug;152(4):1363-72. 

Control of ribosomal protein L1 synthesis in mesophilic and thermophilic
archaea.

Kraft A, Lutz C, Lingenhel A, Grobner P, Piendl W.

Institute of Medical Chemistry and Biochemistry, University of Innsbruck, A-6020
Innsbruck, Austria.

The mechanisms for the control of ribosomal protein synthesis have been
characterized in detail in Eukarya and in Bacteria. In Archaea, only the
regulation of the MvaL1 operon (encoding ribosomal proteins MvaL1, MvaL10, and
MvaL12) of the mesophilic Methanococcus vannielii has been extensively
investigated. As in Bacteria, regulation takes place at the level of
translation. The regulator protein MvaL1 binds preferentially to its binding
site on the 23S rRNA, and, when in excess, binds to the regulatory target site
on its mRNA and thus inhibits translation of all three cistrons of the operon.
The regulatory binding site on the mRNA, a structural mimic of the respective
binding site on the 23S rRNA, is located within the structural gene about 30
nucleotides downstream of the ATG start codon. MvaL1 blocks a step before or at
the formation of the first peptide bond of MvaL1. Here we demonstrate that a
similar regulatory mechanism exists in the thermophilic M. thermolithotrophicus
and M. jannaschii. The L1 gene is cotranscribed together with the L10 and L11
gene, in all genera of the Euryarchaeota branch of the Archaea studied so far. A
potential regulatory L1 binding site located within the structural gene, as in
Methanococcus, was found in Methanobacterium thermoautotrophicum and in
Pyrococcus horikoshii. In contrast, in Archaeoglobus fulgidus a typical L1
binding site is located in the untranslated leader of the L1 gene as described
for the halophilic Archaea. In Sulfolobus, a member of the Crenarchaeota, the L1
gene is part of a long transcript (encoding SecE, NusG, L11, L1, L10, L12). A
previously suggested regulatory L1 target site located within the L11 structural
gene could not be confirmed as an L1 binding site.

PMID: 10430567 [PubMed - indexed for MEDLINE]



NR62: J Virol. 1999 Aug;73(8):7070-6. 

Resistance of ribosomal protein mRNA translation to protein synthesis shutoff
induced by poliovirus.

Cardinali B, Fiore L, Campioni N, De Dominicis A, Pierandrei-Amaldi P.

Istituto di Biologia Cellulare CNR, Istituto Superiore di Sanita', Rome, Italy.

Poliovirus infection induces an overall inhibition of host protein synthesis,
although some mRNAs continue to be translated, suggesting different translation
requirements for cellular mRNAs. It is known that ribosomal protein mRNAs are
translationally regulated and that the phosphorylation of ribosomal protein S6
is involved in the regulation. Here, we report that the translation of ribosomal
protein mRNAs resists poliovirus infection and correlates with an increase in
p70(s6k) activity and phosphorylation of ribosomal protein S6.

PMID: 10400812 [PubMed - indexed for MEDLINE]



NR63: J Mol Biol. 1999 Jun 4;289(2):223-33. 

Mapping the ribosomal RNA neighborhood of protein L11 by directed hydroxyl
radical probing.

Holmberg L, Noller HF.

Center for the Molecular Biology of RNA, Sinsheimer Laboratories, University of
California, Santa Cruz, CA, 95064, USA.

Ribosomal protein L11 is a highly conserved protein that has been implicated in
binding of elongation factors to ribosomes and associated GTP hydrolysis. Here,
we have analyzed the ribosomal RNA neighborhood of Escherichia coli L11 in 50 S
subunits by directed hydroxyl radical probing from Fe(II) tethered to five
engineered cysteine residues at positions 19, 84, 85, 92 and 116 via the linker
1-(p -bromoacetamidobenzyl)-EDTA. Correct assembly of the L11 derivatives was
analyzed by incorporating the modified proteins into 50 S subunits isolated from
an E. coli strain that lacks L11 and testing for previously characterized
L11-dependent footprints in domain II of 23 S rRNA. Hydroxyl radicals were
generated from Fe(II) tethered to L11 and sites of cleavage in the ribosomal RNA
were detected by primer extension. Strong cleavages were detected within the
previously described binding site of L11, in the 1100 region of 23 S rRNA.
Moreover, Fe(II) tethered to position 19 in L11 targeted the backbone of the
sarcin loop in domain VI while probing from position 92 cleaved the backbone
around bases 900 and 2470 in domains II and V, respectively. Fe(II) tethered to
positions 84, 85 and 92 also generated cleavages in 5 S rRNA around helix II.
These data provide new information about the positions of specific features of
23 S rRNA and 5 S rRNA relative to protein L11 in the 50 S subunit and show that
L11 is near highly conserved elements of the rRNA that have been implicated in
binding of tRNA and elongation factors to the ribosome. Copyright 1999 Academic
Press.

PMID: 10366501 [PubMed - indexed for MEDLINE]



DR64: Biochemistry. 1999 Jun 1;38(22):7111-7. 

Human bleomycin hydrolase binds ribosomal proteins.

Koldamova RP, Lefterov IM, DiSabella MT, Almonte C, Watkins SC, Lazo JS.

Department of Pharmacology, University of Pittsburgh School of Medicine,
Pennsylvania 15261, USA.

Bleomycin hydrolase (BH) is a cysteine proteinase that inactivates the
anticancer drug bleomycin. Yeast BH forms a homohexameric structure that
resembles a 20S proteasome and binds to single-stranded RNA and DNA. We now
demonstrate that human BH (hBH) interacts and colocalizes with ribosomal
proteins. Using a yeast two-hybrid system, we found hBH bound to human
homologues of rat ribosomal proteins L11 and L29. The N-terminus of hBH (amino
acids 14-175), which contains a catalytic Cys93, was critical for the binding to
L11 in the two-hybrid environment. hBH precipitated 35S-labeled L11 and L29 in
vitro, and hBH colocalized with L11 and L29 as determined by immunofluorescence.
In addition to cytosolic bleomycin hydrolase, we found abundant bleomycin
hydrolase activity associated with the ribosomal subcellular fraction by
differential centrifugation. hBH was also detected by Western immunoblotting in
a high-speed particulate fraction, where the majority of L11 and L29 were found.
In vitro experiments showed recombinant hBH binds to Chinese hamster ovary cell
microsomes. Thus, our data strongly suggest that hBH exists as both a free
cytosolic and ribosome-associated protein.

PMID: 10353821 [PubMed - indexed for MEDLINE]



NR65: Cytogenet Cell Genet. 1999;84(1-2):97-8. 

Assignment of the L11 ribosomal protein gene (RPL11) to human chromosome
1p36.1-->p35 by in situ hybridization.

Graphodatsky AS, Vorobieva NV, Filipenko ML, Voronina EV, Frengen E, Prydz H.

Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk,
Russia. graf@bionet.nsc.ru

PMID: 10343117 [PubMed - indexed for MEDLINE]



NR66: Cell. 1999 May 14;97(4):491-502. 

Comment in:
    Cell. 1999 May 14;97(4):423-6.

A detailed view of a ribosomal active site: the structure of the L11-RNA complex.

Wimberly BT, Guymon R, McCutcheon JP, White SW, Ramakrishnan V.

Department of Biochemistry, University of Utah School of Medicine, Salt Lake
City 84132, USA.

We report the crystal structure of a 58 nucleotide fragment of 23S ribosomal RNA
bound to ribosomal protein L11. This highly conserved ribonucleoprotein domain
is the target for the thiostrepton family of antibiotics that disrupt elongation
factor function. The highly compact RNA has both familiar and novel structural
motifs. While the C-terminal domain of L11 binds RNA tightly, the N-terminal
domain makes only limited contacts with RNA and is proposed to function as a
switch that reversibly associates with an adjacent region of RNA. The sites of
mutations conferring resistance to thiostrepton and micrococcin line a narrow
cleft between the RNA and the N-terminal domain. These antibiotics are proposed
to bind in this cleft, locking the putative switch and interfering with the
function of elongation factors.

PMID: 10338213 [PubMed - indexed for MEDLINE]



NR67: Cell. 1999 May 14;97(4):423-6. 

Comment on:
    Cell. 1999 May 14;97(4):491-502.

Ribosomal mechanics, antibiotics, and GTP hydrolysis.

Porse BT, Garrett RA.

RNA Regulation Centre, Institute of Molecular Biology, Copenhagen University,
Copenhagen K, Denmark.

Publication Types:
    Comment
    Review
    Review, Tutorial

PMID: 10338205 [PubMed - indexed for MEDLINE]



NR68: J Protein Chem. 1999 Feb;18(2):215-23. 

Structural and functional studies on the overproduced L11 protein from Thermus
thermophilus.

Triantafillidou D, Simitsopoulou M, Franceschi F, Choli-Papadopoulou T.

Laboratory of Biochemistry, School of Chemistry, Aristotle University of
Thessaloniki, Greece.

The L11 ribosomal protein from Thermus thermophilus (TthL11) has been
overproduced and purified to homogeneity using a two-step purification protocol.
The overproduced protein carries a similar methylation pattern at Lys-3 as does
its homolog from Escherichia coli. Chymotrypsin digested only a small part of
the TthL11 protein and did not cleave TthL11 into two peptides, as in the case
of EcoL11, but produced only a single N-terminal peptide. Tryptic digestion of
TthL11 also produced an N-terminal peptide, in contrast to the C-terminal
peptide obtained with L11 from Bacillus stearothermophilus. The recombinant
protein forms a specific complex with a 55-nt 23S rRNA fragment known to
interact with members of the L11 family from several organisms. Cooperative
binding of TthL11 and thiostrepton to 23S rRNA leads to an increased protection
of TthL11 from tryptic digestion. The similar structural and biochemical
properties as well as the significant homology between L11 from E. coli and B.
stearothermophilus with the corresponding protein from Thermus thermophilus
indicate an evolutionarily conserved protein important for ribosome function.

PMID: 10333296 [PubMed - indexed for MEDLINE]



NR69: Science. 1999 May 14;284(5417):1171-4. 

Crystal structure of a conserved ribosomal protein-RNA complex.

Conn GL, Draper DE, Lattman EE, Gittis AG.

Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.

The structure of a highly conserved complex between a 58-nucleotide domain of
large subunit ribosomal RNA and the RNA-binding domain of ribosomal protein L11
has been solved at 2.8 angstrom resolution. It reveals a precisely folded RNA
structure that is stabilized by extensive tertiary contacts and contains an
unusually large core of stacked bases. A bulge loop base from one hairpin of the
RNA is intercalated into the distorted major groove of another helix; the
protein locks this tertiary interaction into place by binding to the
intercalated base from the minor groove side. This direct interaction with a key
ribosomal RNA tertiary interaction suggests that part of the role of L11 is to
stabilize an unusual RNA fold within the ribosome.

PMID: 10325228 [PubMed - indexed for MEDLINE]



NR70: Biochemistry. 1999 Mar 23;38(12):3633-40. 

Protein-RNA sequence covariation in a ribosomal protein-rRNA complex.

GuhaThakurta D, Draper DE.

Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218,
USA.

Comparative sequence analysis has successfully predicted secondary structure and
tertiary interactions in ribosomal and other RNAs. Experiments presented here
ask whether the scope of comparative sequence-based predictions can be extended
to specific interactions between proteins and RNA, using as a system the
well-characterized C-terminal RNA binding domain of ribosomal protein L11
(L11-C76) and its 58 nucleotide binding region in 23S rRNA. The surface of
L11-C76 alpha-helix 3 is known to contact RNA; position 69 in this helix is
conserved as serine in most organisms but varies to asparagine (all plastids) or
glutamine (Mycoplasma). RNA sequence substitutions unique to these groups of
organisms occur at base pairs 1062/1076 or 1058/1080, respectively. The
possibility that rRNA base pair substitutions compensate for variants in L11
alpha-helix 3 has been tested by measuring binding affinities between sets of
protein and RNA sequence variants. Stability of the RNA tertiary structure, as
measured by UV melting experiments, was unexpectedly affected by a 1062/1076
base pair substitution; additional mutations were required to restore a stably
folded structure to this RNA. The results show that the asparagine variant of
L11-C76 residue 69 has been compensated by substitution of a 1062/1076 base
pair, and plausibly suggest a direct contact between the amino acid and base
pair. For some of the protein and RNA mutations studied, changes in binding
affinity probably reflect longer-range adjustments of the protein-RNA contact
surface.

PMID: 10090750 [PubMed - indexed for MEDLINE]



NR71: J Mol Biol. 1999 Mar 19;287(1):33-45. 

The antibiotic micrococcin acts on protein L11 at the ribosomal GTPase centre.

Porse BT, Cundliffe E, Garrett RA.

RNA Regulation Centre Institute of Molecular Biology, University of Copenhagen,
Solvgade 83H, Copenhagen K, DK1307, Denmark.

Micrococcin-resistant mutants of Bacillus megaterium that carry mutations
affecting ribosomal protein L11 have been characterised. The mutants fall into
two groups. "L11-minus" strains containing an L11 gene with deletions,
insertions or nonsense mutations which grow 2.5-fold slower than the wild-type
strain, whereas other mutants carrying single-site substitutions within an 11
amino acid residue segment of the N-terminal domain of L11 grow normally.
Protein L11 binds to 23 S rRNA within the ribosomal GTPase centre which
regulates GTP hydrolysis on ribosomal factors. Micrococcin binding within the
rRNA component of this centre was probed on wild-type and mutant ribosomes, in
vivo, using dimethyl sulphate where it generated an rRNA footprint
indistinguishable from that produced in vitro, even after the cell growth had
been arrested by treatment with either kirromycin or fusidic acid. No drug-rRNA
binding was detected in vivo for the L11-minus mutants, while reduced binding
(approximately 30-fold) was observed for two single-site mutants P23L and P26L.
For the latter, the reduced drug affinity alone did not account for the
resistance-phenotype because rapid cell growth occurred even at drug
concentrations that would saturate the ribosomes. Micrococcin was also bound to
complexes containing an rRNA fragment and wild-type or mutant L11, expressed as
fusion proteins, and they were probed with proteinases. The drug produced strong
protection effects on the wild-type protein and weak effects on the P23L and
P26L mutant proteins. We infer that inhibition of cell growth by micrococcin, as
for thiostrepton, results from the imposition of a conformational constraint on
protein L11 which, in turn, perturbs the function(s) of the ribosomal
factor-guanosine nucleotide complexes. Copyright 1999 Academic Press.

PMID: 10074405 [PubMed - indexed for MEDLINE]



NR72: J Antibiot (Tokyo). 1998 Oct;51(10):954-7. 

Sequence analysis of the ribosomal L11 protein gene (rplK=relC) in Streptomyces
lavendulae using a deletion allele.

Kawamoto S, Zhang D, Ochi K.

National Food Research Institute, Tsukuba, Ibaraki, Japan.

PMID: 9917010 [PubMed - indexed for MEDLINE]



NR73: Dev Genes Evol. 1999 Jan;209(1):63-8. 

Green fluorescent proteins with short half-lives as reporters in Dictyostelium
discoideum.

Deichsel H, Friedel S, Detterbeck A, Coyne C, Hamker U, MacWilliams HK.

Zoologisches Institut, Ludwig-Maximilians-Universitat, Luisenstrasse 14, D-80333
Munchen, Germany.

We describe two modifications of the popular reporter green fluorescent protein
(GFP) which have short half-lives in our system, the cellular slime mould
Dictyostelium discoideum. One of these bears an N-terminal ubiquitin; this GFP
was originally planned to be a substrate of the "N-end-rule" pathway, but
deubiquitination does not seem to occur, and a degradation by the UFD
(ubiquitin-fusion-degradation pathway seems more probable. The protein half-life
is about 3-5 h. The second construct has an N-terminus derived from the L11
ribosomal protein; it is transported to the nucleus and broken down much more
rapidly than the ubiquitin fusion (protein half-life about 30 min). We show
examples of the use of these reporters in the study of gene expression in
Dictyostelium.

PMID: 9914420 [PubMed - indexed for MEDLINE]



NR74: Nucleic Acids Res. 1999 Jan 15;27(2):381-8. 

RNA binding strategies of ribosomal proteins.

Draper DE, Reynaldo LP.

Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.
draper@jhunix.hcf.jhu.edu

Structures of a number of ribosomal proteins have now been determined by
crystallography and NMR, though the complete structure of a ribosomal
protein-rRNA complex has yet to be solved. However, some ribosomal protein
structures show strong similarity to well-known families of DNA or RNA binding
proteins for which structures in complex with cognate nucleic acids are
available. Comparison of the known nucleic acid binding mechanisms of these
non-ribosomal proteins with the most highly conserved surfaces of similar
ribosomal proteins suggests ways in which the ribosomal proteins may be binding
RNA. Three binding motifs, found in four ribosomal proteins so far, are
considered here: homeodomain-like alpha-helical proteins (L11), OB fold proteins
(S1 and S17) and RNP consensus proteins (S6). These comparisons suggest that
ribosomal proteins combine a small number of fundamental strategies to develop
highly specific RNA recognition sites.

Publication Types:
    Review

PMID: 9862955 [PubMed - indexed for MEDLINE]



NR75: Int J Syst Bacteriol. 1998 Oct;48 Pt 4:1187-96. 

Comment in:
    Int J Syst Evol Microbiol. 2003 Sep;53(Pt 5):1697-8.

Proposal of a new halobacterial genus Natrinema gen. nov., with two species
Natrinema pellirubrum nom. nov. and Natrinema pallidum nom. nov.

McGenity TJ, Gemmell RT, Grant WD.

Postgraduate Research Institute for Sedimentology, University of Reading,
Whiteknights, UK. t.j.mcgenity@reading.ac.uk

A phylogenetic analysis of 69 halobacterial 16S rRNA gene sequences has been
carried out, integrating data from new isolates, previously described
halobacteria and cloned sequences from uncultivated halobacteria. Halobacterium
halobium NCIMB 777, Halobacterium trapanicum NCIMB 784 and Halobacterium
salinarium NCIMB 786, together with several other strains (strains T5.7, L11 and
Halobacterium trapanicum NCIMB 767) constitute a distinct lineage with at least
98.2% sequence similarity. These strains have been incorrectly assigned to the
genus Halobacterium. Therefore, based on a variety of taxonomic criteria, it is
proposed that Halobacterium salinarium NCIMB 786 is renamed as Natrinema
pellirubrum nom. nov., the type species of the new genus Natrinema gen. nov.,
and that Halobacterium halobium NCIMB 777 and Halobacterium trapanicum NCIMB 784
are renamed as a single species, Natrinema pallidum nom. nov. It was notable
that halobacteria closely related to the proposed new genus have been isolated
from relatively low-salt environments.

PMID: 9828420 [PubMed - indexed for MEDLINE]



NR76: Int J Syst Bacteriol. 1998 Apr;48 Pt 2:597-600. 

Comparative ribosomal protein (L11 and L30) sequence analyses of several
Streptomyces spp. commonly used in genetic studies.

Kawamoto S, Ochi K.

National Food Research Institute, Ibaraki, Japan.

The taxonomic relationships among nine strains of Streptomyces, which have been
commonly used for genetic studies, were examined by sequence analysis of their
ribosomal L11(= rplK) protein genes. Phylogenetic relationships among these
organisms derived from similarity sequence analysis of the rplK genes were in
good agreement with those derived from the analysis of the deduced L11 protein
amino acid sequence itself, indicating complete sequence homology among
Streptomyces coelicolor A3(2), 'Streptomyces lividans 66' and Streptomyces
violaceoruber JCM 4423. S. coelicolor A3(2) related (in the order of closer
relatedness) to Streptomyces antibioticus ATCC 14888, Streptomyces griseus IFO
13189, Streptomyces lavendulae MA 406 A-1 and Streptomyces virginiae MAFF 6014.
Sequence analysis of the 26 N-terminal amino acid residues of ribosomal L30
proteins also resulted in similar phylogenetic relationships, except that S.
griseus, S. lavendulae and S. virginiae were not differentiated from each other
using this method. These findings concerning the phylogenetic relationship
therefore confirm the previous conclusion that S. coelicolor A3(2), 'S. lividans
66' and S. violaceoruber should be recognized as a single taxon at the species
level.

PMID: 9731302 [PubMed - indexed for MEDLINE]



NR77: J Protein Chem. 1998 Aug;17(6):519-20. 

Thermus thermophilus L11 ribosomal protein: cloning, overproduction, structural,
and functional studies--in vivo methylation of the overproduced protein.

Triantafillidou D, Simitsopoulou M, Franceschi F, Choli-Papadopoulou T.

Laboratory of Biochemistry, School of Chemistry, Aristotle University of
Thessaloniki, Greece.

PMID: 9723724 [PubMed - indexed for MEDLINE]



NR78: Biochemistry. 1998 Aug 25;37(34):11980-8. 

A functional ribosomal RNA tertiary structure involves a base triple
interaction.

Conn GL, Gutell RR, Draper DE.

Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland
21218, USA.

Comparative sequence analysis reveals a coordinated set of nucleotide exchanges
between the base pair 1092/1099 and the unpaired position 1072 [(1092/1099)1072]
in the L11 binding domain of 23S ribosomal RNA. This set of exchanges has
occurred at least 4 times during evolution, suggesting that these positions form
a base triple. The analysis further suggests an important role for positions
(1065/1073), adjacent to 1072. The covariation at positions (1092/1099)1072 is
studied here by analysis of RNA variants using UV melting and binding of
ribosomal protein L11 and thiostrepton to assay for tertiary folding of this
domain. The tertiary structure of the RNA is eliminated by alteration of the
unpaired nucleotide (C1072 to U mutation), and binding of L11 and thiostrepton
are reduced 10-fold compared to the wild type. In contrast, substitution of the
base pair (CG1092/1099 to UA mutation) allows formation of the tertiary
structure but dramatically alters the pH dependence of tertiary folding. The
fully compensated set of mutations, (CG)C to (UA)U, restores the tertiary
structure of the RNA to a state almost identical to the wild type. The nature of
this base triple and its implications for the folding of the RNA and ligand
interactions are discussed.

PMID: 9718323 [PubMed - indexed for MEDLINE]



NR79: EMBO J. 1998 Aug 17;17(16):4559-71. 

The solution structure of ribosomal protein S4 delta41 reveals two subdomains
and a positively charged surface that may interact with RNA.

Markus MA, Gerstner RB, Draper DE, Torchia DA.

Molecular Structural Biology Unit, National Institute of Dental Research,
National Institutes of Health, 30 Convent Drive, Room 132, Bethesda, MD
20892-4320, USA.

S4 is one of the first proteins to bind to 16S RNA during assembly of the
prokaryotic ribosome. Residues 43-200 of S4 from Bacillus stearothermophilus (S4
Delta41) bind specifically to both 16S rRNA and to a pseudoknot within the alpha
operon mRNA. As a first step toward understanding how S4 recognizes and
organizes RNA, we have solved the structure of S4 Delta41 in solution by
multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The fold
consists of two globular subdomains, one comprised of four helices and the other
comprised of a five-stranded antiparallel beta-sheet and three helices. Although
cross-linking studies suggest that residues between helices alpha2 and alpha3
are close to RNA, the concentration of positive charge along the crevice between
the two subdomains suggests that this could be an RNA-binding site. In contrast
to the L11 RNA-binding domain studied previously, S4 Delta41 shows no fast local
motions, suggesting that it has less capacity for refolding to fit RNA. The
independently determined crystal structure of S4 Delta41 shows similar features,
although there is small rotation of the subdomains compared with the solution
structure. The relative orientation of the subdomains in solution will be
verified with further study.

PMID: 9707416 [PubMed - indexed for MEDLINE]



NR80: Biol Chem. 1998 Jul;379(7):857-66. 

Efficient in vitro translational termination in Escherichia coli is constrained
by the orientations of the release factor, stop signal and peptidyl-tRNA within
the termination complex.

McCaughan KK, Poole ES, Pel HJ, Mansell JB, Mannering SA, Tate WP.

Department of Biochemistry and Centre for Gene Research, University of Otago,
Dunedin, New Zealand.

There have been contrasting reports of whether the positioning of a
translational stop signal immediately after a start codon in a single
oligonucleotide can act as a model template to support efficient in vitro
termination. This paradox stimulated this study of what determines the
constraints on the positioning of the components in the termination complex. The
mini mRNA, AUGUGAA, was unable to support efficient in vitro termination in
contrast to separate AUG/UGA(A) codons, unless the ribosomal interaction of the
stop signal with the decoding factor, release factor 2, was stimulated with
ethanol or with nucleotide-free release factor 3, or by using (L11-)-ribosomes
which have a higher affinity for release factor 2, or unless the fMet-tRNA was
first bound to 30S subunits independently of the mini mRNA. An additional
triplet stop codon could restore activity of the mini mRNA, indicating that its
recognition was not sterically restrained by the stop signal already within it.
This suggests that in an initiation complex an adjoining start/stop signal is
not positioned to support efficient decoding by release factor unless it is
separated from the start codon. Site-directed crosslinking from mRNAs to
components of the termination complex has shown that mRNA elements like the
Shine-Dalgarno sequence and the codon preceding the stop signal can affect the
crosslinking to release factor, and presumably the orientation of the signal to
the factor.

PMID: 9705149 [PubMed - indexed for MEDLINE]



NR81: Biol Chem. 1998 Jul;379(7):819-29. 

A novel cell-free system for peptide synthesis driven by pyridine.

Nitta I, Nambu H, Okado T, Yoshinari S, Ueda T, Endo Y, Nierhaus KH, Watanabe K.

Department of Chemistry & Biotechnology, Graduate School of Engineering,
University of Tokyo, Japan.

Previously we demonstrated that ribosomes can synthesize polypeptides in the
presence of high concentrations (40-60%) of pyridine without any protein
factors. Here we analyze additional ribosomal parameters in 60% pyridine using
Escherichia coli ribosomes. Ribosomal subunits once exposed to pyridine failed
to re-associate to 70S ribosomes in aqueous buffer systems even in the presence
of 20 mM Mg2+, whereas they formed 70S complexes in the presence of 60%
pyridine. Two-dimensional gel electrophoresis of ribosomal proteins revealed
that some proteins located at the protuberances of the large subunit, e. g.
L7/L12 and L11 forming the elongation factor-binding domain, were released in
the pyridine system. The aminoglycoside neomycin, a strong inhibitor of the
ribosomal (factor-independent) translocation reaction, completely blocked
poly(Phe) synthesis and translocation activities in the pyridine system, whereas
these activities were not affected at all by gypsophilin, a ribotoxin that
inhibits factor-dependent translocation. Another inhibitor of the ribosomal
translocation, thiostrepton, had no effect concerning the two activities, which
is consistent with the fact that this antibiotic requires L11 for its binding to
the ribosome. These results suggest that the ribosomes can perform a
translocation reaction in the pyridine system, but in a factor-independent
(spontaneous) manner.

PMID: 9705145 [PubMed - indexed for MEDLINE]



NR82: Curr Opin Struct Biol. 1998 Jun;8(3):294-300. 

Structure and dynamics of ribosomal RNA.

Woodson SA, Leontis NB.

Department of Chemistry and Biochemistry, University of Maryland, College Park
20742-2021, USA. sw74@umail.umd.edu

Over the past two years, progress in X-ray crystallography, NMR spectroscopy and
electron microscopy has begun to reveal the complex structure of the RNA within
the ribosome. The structures of ribosomal proteins L11 and S15, among others,
show how RNA-protein interactions organize the conformation of the junctions
between ribosomal RNA helices. Genetic and biochemical methods have also
identified a three base-pair switch within the 16S rRNA that is linked to mRNA
decoding.

Publication Types:
    Review
    Review, Tutorial

PMID: 9666324 [PubMed - indexed for MEDLINE]



NR83: Curr Genet. 1998 Apr;33(4):304-10. 

A ribosomal protein gene cluster is encoded in the mitochondrial DNA of
Dictyostelium discoideum: UGA termination codons and similarity of gene order to
Acanthamoeba castellanii.

Iwamoto M, Pi M, Kurihara M, Morio T, Tanaka Y.

Institute of Biological Sciences, University of Tsukuba, Japan.

We sequenced a region of about 14.5 kb downstream from the ribosomal protein L11
gene (rpl11) in the mitochondrial DNA (54+/-2 kb) of the cellular slime mold
Dictyostelium discoideum. Sequence analysis revealed that eleven ribosomal
protein genes and six open reading frames (ORFs) formed a cluster arranged in
the order: rpl11-orf189-rps12-rps7-rpl2-rps19-+
++orf425-orf1740-rpl16-rpl14-orf188- rps14-rps8-rpl6-rps13-orf127-orf796. This
order was very similar to that of homologous genes in Acanthamoeba castellanii
mitochondrial DNA. The N-terminal region of ORF425 and the C-terminal region of
ORF1740 had partial similarities to the S3 ribosomal protein of other organisms.
The termination codons of rpl16 and orf188 were UGA, which has not hitherto been
found in genes encoded in D. discoideum mitochondrial DNA.

PMID: 9560439 [PubMed - indexed for MEDLINE]



NR84: J Mol Biol. 1998 Feb 20;276(2):391-404. 

The antibiotic thiostrepton inhibits a functional transition within protein L11
at the ribosomal GTPase centre.

Porse BT, Leviev I, Mankin AS, Garrett RA.

RNA Regulation Centre, University of Copenhagen, Denmark.

A newly identified class of highly thiostrepton-resistant mutants of the
archaeon Halobacterium halobium carry a missense mutation at codon 18 within the
gene encoding ribosomal protein L11. In the mutant proteins, a proline,
conserved in archaea and bacteria, is converted to either serine or threonine.
The mutations do not impair either the assembly of the mutant L11 into 70 S
ribosomes in vivo or the binding of thiostrepton to ribosomes in vitro.
Moreover, the corresponding mutations at proline 22, in a fusion protein of L11
from Escherichia coli with glutathione-S-transferase, did not reduce the binding
affinities of the mutated L11 fusion proteins for rRNA of of thiostrepton for
the mutant L11-rRNA complexes at rRNA concentrations lower than those prevailing
in vivo. Probing the structure of the fusion protein of wild-type L11, from E.
coli, using a recently developed protein footprinting technique, demonstrated
that a general tightening of the C-terminal domain occurred on rRNA binding,
while thiostrepton produced a footprint centred on tyrosine 62 at the junction
of the N and C-terminal domains of protein L11 complexed to rRNA. The intensity
of this protein footprint was strongly reduced for the mutant L11-rRNA
complexes. These results indicate that although, as shown earlier, thiostrepton
binds primarily to 23 S rRNA, the drug probably inhibits peptide elongation by
impeding a conformational change within protein L11 that is important for the
function of the ribosomal GTPase centre. This putative inhibitory mechanism of
thiostrepton is critically dependent on proline 18/22. Moreover, the absence of
this proline from eukaryotic protein L11 sequences would account for the high
thiostrepton resistance of eukaryotic ribosomes.

PMID: 9512711 [PubMed - indexed for MEDLINE]



NR85: Mol Gen Genet. 1998 Jan;257(2):219-29. 

Cloning and transcriptional analysis of the rplKA-or f31-rplJL gene cluster of
Streptomyces griseus.

Kuster C, Piepersberg W, Distler J.

Institut fur Chemische Mikrobiologie, Bergische Universitat GH Wuppertal,
Germany.

A 5018-bp DNA fragment of the rpl/rpo BC gene cluster (here called the rif
cluster) of Streptomyces griseus N2-3-11 was analysed by DNA sequencing and
transcription studies. By sequence comparison of the deduced proteins, five
genes and part of an open reading frame (orf) were identified. The genes
encoding the ribosomal (r-) proteins L1 (rplA), L7/12 (rplJ), L10 (rplK) and L11
(rplL), a protein of known function (orf31), and the N-terminus of the beta
subunit of RNA polymerase (rpoB), are organized in three operons, rplKA, rplJL
and rpoB(C), and the monocistronic transcription unit orf31. The promoters of
these transcription units, rplKp, orf31p, rplJp, and rpoBp, were identified and
the growth-phase dependence of the transcription of these operons was analysed.
Binding sites for the ribosomal proteins L1 and L10 were identified by sequence
comparison, suggesting that the r-proteins RplA and RplJ are involved in
feedback regulation of their respective operons by binding to specific
RNA-binding sites present in both the mRNA and the 23S rRNA, as has been
described for other bacteria. The analyses of the rpoBp promoter by means of
promoter-probe plasmids suggested a possible attenuator-based regulatory
mechanism for the transcription of the rpoB(C) operon.

PMID: 9491081 [PubMed - indexed for MEDLINE]



NR86: Mol Microbiol. 1998 Jan;27(2):455-68. 

MvaL1 autoregulates the synthesis of the three ribosomal proteins encoded on the
MvaL1 operon of the archaeon Methanococcus vannielii by inhibiting its own
translation before or at the formation of the first peptide bond.

Mayer C, Kohrer C, Grobner P, Piendl W.

Institut fur Medizinische Chemie und Biochemie, Universitat Innsbruck, Austria.

The control of ribosomal protein synthesis has been investigated extensively in
Eukarya and Bacteria. In Archaea, only the regulation of the MvaL1 operon
(encoding ribosomal proteins MvaL1, MvaL10 and MvaL12) of Methanococcus
vannielii has been studied in some detail. As in Escherichia coil, regulation
takes place at the level of translation. MvaL1, the homologue of the regulatory
protein L1 encoded by the L11 operon of E. coli, was shown to be an
autoregulator of the MvaL1 operon. The regulatory MvaL1 binding site on the mRNA
is located about 30 nucleotides downstream of the ATG start codon, a sequence
that is not in direct contact with the initiating ribosome. Here, we demonstrate
that autoregulation of MvaL1 occurs at or before the formation of the first
peptide bond of MvaL1. Specific interaction of purified MvaL1 with both 23S RNA
and its own mRNA is confirmed by filter binding studies. In vivo expression
experiments reveal that translation of the distal MvaL10 and MvaL12 cistrons is
coupled to that of the MvaL1 cistron. A mRNA secondary structure resembling a
canonical L10 binding site and preliminary in vitro regulation experiments had
suggested a co-regulatory function of MvaL10, the homologue of the regulatory
protein L10 of the beta-operon of E. coil. However, we show that MvaL10 does not
have a regulatory function.

PMID: 9484899 [PubMed - indexed for MEDLINE]



NR87: J Biol Chem. 1998 Jan 16;273(3):1670-6. 

Cross-linking of selected residues in the N- and C-terminal domains of
Escherichia coli protein L7/L12 to other ribosomal proteins and the effect of
elongation factor Tu.

Dey D, Bochkariov DE, Jokhadze GG, Traut RR.

Department of Biological Chemistry, School of Medicine, University of
California, Davis 95616, USA.

Five different variants of protein L7/L12, each with a single cysteine
substitution at a selected site, were produced, modified with
125I-N-[4-(p-azidosalicylamido)-butyl]-3-(2'-pyridyldithio)propion amide, a
radiolabeled, sulfhydryl-specific, heterobifunctional, cleavable
photocross-linking reagent that transfers radiolabel to the target molecule upon
reduction of the disulfide bond. The proteins were reconstituted with core
particles depleted of wild type L7/L12 to yield 70 S ribosomes. Cross-linked
molecules were identified and quantified by the radiolabel. No cross-linking of
RNA was detected. Two sites in the dimeric N-terminal domain, Cys-12 and Cys-33,
cross-linked strongly to L10 and in lower yield to L11 but to no other proteins.
The three sites in the globular C-terminal domain all cross-linked strongly to
L11 and, in lower yield, to L10. Weaker cross-linking to 50 S proteins L2 and L5
occurred from all three C-terminal domain locations. The 30 S ribosomal proteins
S2, S3, S7, S14, S18 were also cross-linked from all three of these sites.
Binding of the ternary complex [14C]Phe-tRNA-elongation factor Tu.guanyl-5'-yl
imidodiphosphate) but not [14C]Phe-tRNA.elongation factor Tu.GDP.kirromycin
increased labeling of L2, L5, and all of the 30 S proteins. These results imply
the flexibility of L7/L12 and the transient proximity of three surfaces of the
C-terminal domain with the base of the stalk, the peptidyl transferase domain,
and the head of the 30 S subunit.

PMID: 9430711 [PubMed - indexed for MEDLINE]



NR88: J Mol Biol. 1997 Nov 21;274(1):101-13. 

The RNA binding domain of ribosomal protein L11: three-dimensional structure of
the RNA-bound form of the protein and its interaction with 23 S rRNA.

Hinck AP, Markus MA, Huang S, Grzesiek S, Kustonovich I, Draper DE, Torchia DA.

National Institute of Dental Research, Bethesda, MD 20892-4326, USA.

The three-dimensional solution structure has been determined by NMR spectroscopy
of the 75 residue C-terminal domain of ribosomal protein L11 (L11-C76) in its
RNA-bound state. L11-C76 recognizes and binds tightly to a highly conserved 58
nucleotide domain of 23 S ribosomal RNA, whose secondary structure consists of
three helical stems and a central junction loop. The NMR data reveal that the
conserved structural core of the protein, which consists of a bundle of three
alpha-helices and a two-stranded parallel beta-sheet four residues in length, is
nearly the same as the solution structure determined for the non-liganded form
of the protein. There are however, substantial chemical shift perturbations
which accompany RNA binding, the largest of which map onto an extended loop
which bridges the C-terminal end of alpha-helix 1 and the first strand of
parallel beta-sheet. Substantial shift perturbations are also observed in the
N-terminal end of alpha-helix 1, the intervening loop that bridges helices 2 and
3, and alpha-helix 3. The four contact regions identified by the shift
perturbation data also displayed protein-RNA NOEs, as identified by
isotope-filtered three-dimensional NOE spectroscopy. The shift perturbation and
NOE data not only implicate helix 3 as playing an important role in RNA binding,
but also indicate that regions flanking helix 3 are involved as well. Loop 1 is
of particular interest as it was found to be flexible and disordered for L11-C76
free in solution, but not in the RNA-bound form of the protein, where it appears
rigid and adopts a specific conformation as a result of its direct contact to
RNA. Copyright 1997 Academic Press Limited.

PMID: 9398519 [PubMed - indexed for MEDLINE]



NR89: J Mol Biol. 1997 Nov 14;273(5):1020-31. 

Affinities and selectivities of divalent cation binding sites within an RNA
tertiary structure.

Bukhman YV, Draper DE.

Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.

A 58 nucleotide fragment of Escherichia coli large subunit ribosomal RNA,
nucleotides 1051 to 1108, adopts a specific tertiary structure normally
requiring both monovalent (NH4+ or K+) and divalent (Mg2+) ions to fold; this
ion-dependent structure is a prerequisite for recognition by ribosomal protein
L11. Melting experiments have been used to show that a sequence variant of this
fragment, GACG RNA, is able to adopt a stable tertiary structure in the presence
of 1.6 M NH4Cl and absence of divalent ions. The similarity of this high-salt
structure to the tertiary structure formed under more typical salt conditions
(0.1 M NH4Cl and several mM MgCl2) was shown by its following properties: (i) an
unusual ratio of hyperchromicity at 260 nm and 280 nm upon unfolding, (ii)
selectivity for NH4+ over K+ or Na+, (iii) stabilization by L11 protein, and
(iv) further stabilization by added Mg2+. Delocalized electrostatic interactions
of divalent ions with nucleic acids should be very weak in the presence of >1 M
monovalent salt; thus stabilization of the tertiary structure by low (<1 mM)
Mg2+ concentrations in these high-salt conditions suggests that Mg2+ binds at
specific site(s). GACG RNA tertiary structure unfolding in 1.6 M NH4Cl (Tm
approximately 39 degrees C) is distinct from melting of the secondary structure
(centered at approximately 72 degrees C), and it has been possible to calculate
the free energy of tertiary structure stabilization upon addition of various
divalent cations. From these binding free energies, ion-RNA binding isotherms
for Mn2+, Mg2+, Ca2+, Sr2+ and Ba2+ have been obtained. All of these ions bind
at two sites: one site favors Mg2+ and Ba2+ and discriminates against Ca2+,
while the other site favors binding of smaller ions over larger ones (Mg2+ >Ca2+
>Sr2+ >Ba2+). Weak cooperative or anticooperative interactions between the
sites, also dependent on ion radius, may also be taking place. Copyright 1997
Academic Press Limited.

PMID: 9367788 [PubMed - indexed for MEDLINE]



NR90: Mol Gen Genet. 1997 Nov;256(5):488-98. 

Molecular and functional analysis of the ribosomal L11 and S12 protein genes
(rplK and rpsL) of Streptomyces coelicolor A3(2).

Ochi K, Zhang D, Kawamoto S, Hesketh A.

National Food Research Institute, Ibaraki, Japan. kochi@ss.nfri.affrc.go.jp

A RelC deletion mutant, KO-100, of Streptomyces coelicolor A3(2) has been
isolated from a collection of spontaneous thiostrepton-resistant mutants. KO-100
grows as vigorously as the parent strain and possesses a 6-bp deletion within
the rplK, previously termed relC. When the wild-type rplK gene was propagated on
a low-copy-number vector in mutant KO-100, the ability to produce ppGpp,
actinorhodin and undecylprodigiosin, which had been lost in the RelC mutant, was
completely restored. Allele replacement by gene homogenotization demonstrated
that the RelC mutation is responsible for the resistance to thiostrepton and the
inactivation of ppGpp, actinorhodin and undecylprodigiosin production. Western
blotting showed that ribosomes from the RelC mutant KO-100 contain only
one-eighth the amount of L11 protein found in ribosomes of the parent strain.
The impairment of antibiotic production in KO-100 could be rescued by the
introduction of mutations that confer resistance to streptomycin (str), which
result in alteration of Lys-88 in ribosomal protein S12 to Glu or Arg. No
accompanying restoration of ppGpp synthesis was detected in these RelC str
double mutants.

PMID: 9413432 [PubMed - indexed for MEDLINE]



NR91: Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12892-7. 

Proteins on ribosome surface: measurements of protein exposure by hot tritium
bombardment technique.

Agafonov DE, Kolb VA, Spirin AS.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow
Region.

The hot tritium bombardment technique [Goldanskii, V. I., Kashirin, I. A.,
Shishkov, A. V., Baratova, L. A. & Grebenshchikov, N. I. (1988) J. Mol. Biol.
201,567-574] has been applied to measure the exposure of proteins on the
ribosomal surface. The technique is based on replacement of hydrogen by high
energy tritium atoms in thin surface layer of macromolecules. Quantitation of
tritium radioactivity of each protein has revealed that proteins S1, S4, S5, S7,
S18, S20, and S21 of the small subunit, and proteins L7/L12, L9, L10, L11, L16,
L17, L24, and L27 of the large subunit are well exposed on the surface of the
Escherichia coli 70 S ribosome. Proteins S8, S10, S12, S16, S17, L14, L20, L29,
L30, L31, L32, L33, and L34 have virtually no groups exposed on the ribosomal
surface. The remaining proteins are found to be exposed to lesser degree than
the well exposed ones. No additional ribosomal proteins was exposed upon
dissociation of ribosomes into subunits, thus indicating the absence of proteins
on intersubunit contacting surfaces.

PMID: 9371771 [PubMed - indexed for MEDLINE]



NR92: Mol Gen Genet. 1997 Aug;255(6):549-60. 

Molecular analysis of the ribosomal L11 protein gene (rplK = relC) of
Streptomyces griseus and identification of a deletion allele.

Kawamoto S, Zhang D, Ochi K.

National Food Research Institute, Ibaraki, Japan.

The rplK (= relC) gene, which codes for ribosomal protein L11, was cloned from
Streptomyces griseus IFO13189 using a screening procedure based on polymerase
chain reaction amplification of a gene segment that subsequently allowed the
isolation of the complete gene from a gene library. rplK lies between the nusG
gene, encoding a protein involved in antitermination of transcription, and the
rplA gene, which encodes the ribosomal protein L1. Comparison of the rplK gene
sequences of the wild-type strain and the presumed relC mutant strain 3-3
(originally isolated as a thiopeptin-resistant isolate) revealed a 12-bp
deletion within the rplK gene from the mutant, flanked by a 4-bp repeat sequence
in the corresponding region in the wild type. When the wild-type rplK gene was
propagated on a low-copy-number vector in the relC mutant 3-3, the ability to
produce guanosine 5'-diphosphate 3'-diphosphate, streptomycin, and submerged
spores was completely restored to parental levels. The impaired ability to form
aerial mycelium was, however, unaffected. Western blotting analysis showed that
the ribosomes from the relC mutant 3-3 incorporate the mutant L11 protein
normally, although the level of incorporation is approximately one-third that of
the wild-type L11 protein in ribosomes of the parent strain. Propagation of the
mutant rplK gene in the wild-type strain resulted in marked defects in growth,
streptomycin production, and aerial mycelium formation, indicating that the
mutant L11 protein exerts certain negative effects in the cells.

PMID: 9323358 [PubMed - indexed for MEDLINE]



NR93: J Bacteriol. 1997 Aug;179(15):4676-83. 

Identification and characterization of an operon of Helicobacter pylori that is
involved in motility and stress adaptation.

Beier D, Spohn G, Rappuoli R, Scarlato V.

Department of Molecular Biology, Immunobiological Research Institute Siena,
Chiron Vaccines, Italy.

We identified a novel stress-responsive operon (sro) of Helicobacter pylori that
contains seven genes which are likely to be involved in cellular functions as
diverse as chemotaxis, heat shock response, ion transport, and posttranslational
protein modification. The products of three of these genes show amino acid
homologies to known proteins, such as the flagellar motor switch protein CheY, a
class of heat shock proteins, and the ribosomal protein L11 methyltransferase,
and to a phosphatidyltransferase. In addition to containing an open reading
frame of unknown function, the product of which is predicted to be membrane
associated, the sro locus contains three open reading frames that have
previously been described as constituting two separate loci, the ftsH gene and
the copAP operon of H. pylori. Knockout mutants showed that CheY is essential
for bacterial motility and that CopA, but not CopP, relieves copper toxicity.
Transcriptional analyses indicated that this locus is regulated by a single
promoter and that a positive effect on transcription is exerted by the addition
of copper to the medium and by temperature upshift from 37 to 45 degrees C. The
possible role of this locus in H. pylori virulence is discussed.

PMID: 9244252 [PubMed - indexed for MEDLINE]



NR94: Biochim Biophys Acta. 1997 Jul 18;1340(2):170-7. 

Nucleotide sequence of a gene cluster encoding NusG and the L11-L1-L10-L12
ribosomal proteins from the thermophilic archaeon Sulfolobus solfataricus.

Geiger M, Grobner P, Piendl W.

Institut fur Medizinische Chemie und Biochemie, Universitat Innsbruck, Austria.

The complete nucleotide sequence of a gene cluster encoding the NusG and the L
11-L1-L10-L12 ribosomal proteins from the thermophilic crenarchaeon Sulfolobus
solfataricus has been determined. The genes are arranged in the same order as
the equivalent genes in the rif region of Escherichia coli. The ribosomal
proteins exhibit between 66% (L10) and 80% (L12) identity with their respective
equivalents from Sulfolobus acidocaldarius. The short distance (5 nucleotides)
between the nusG stop codon and the L11 start codon suggests that nusG and the
genes for the ribosomal proteins are transcribed as a single unit.

PMID: 9252104 [PubMed - indexed for MEDLINE]



NR95: Biochem Biophys Res Commun. 1997 Jul 18;236(2):510-6. 

The eukaryotic translation initiation factor 5, eIF-5, a protein from Zea mays,
containing a zinc-finger structure, binds nucleic acids in a zinc-dependent
manner.

Lopez Ribera I, Ruiz-Avila L, Puigdomenech P.

Departament de Genetica Molecular, CID-CSIC, Jordi Girona, Barcelona, Spain.

A maize cDNA encoding the eukaryotic translation initiation factor 5 (eIF-5) has
been isolated from an 8-day-old seedling cDNA library. The 1975 bp cDNA encodes
a protein of 451 amino acids, with a predicted molecular weight of 49.04 kDa,
and hybridizes to a single sequence in the maize genome. The deduced sequence
contains motifs characteristic of proteins belonging to the GPTase superfamily,
a zinc finger well conserved in all the protein sequences for eIF-5 reported so
far, and a fragment also present in prokaryotic and chloroplast L11 ribosomal
protein. Polymer-binding assays have been used to assess the predicted RNA
binding property of the protein and to characterize its function. It is shown
that the eIF-5-encoded protein binds to single-stranded DNA and to polyuridylic
acid and that the binding is dependent on the presence of Zn2+ ions. These
results suggest that the zinc-finger structure is involved in the binding of the
eIF-5 protein to RNA.

PMID: 9240471 [PubMed - indexed for MEDLINE]



NR96: Curr Genet. 1997 May;31(5):396-400. 

The yeast ORF YDL202w codes for the mitochondrial ribosomal protein YmL11.

Bui DM, Jarosch E, Schweyen RJ.

Vienna Biocenter, Institute of Microbiology and Genetics, University of Vienna,
A-1030 Wien, Austria.

The Saccharomyces cerevisiae open reading frame YDL202w has been characterised
in the course of the EUROFAN yeast genome analysis program. Disruption of
YDL202w causes a respiratory deficient phenotype accompanied by a loss of
mitochondrial DNA. This phenotype is usually found in mutants defective in
mitochondrial replication or gene expression. YDL202w has the potential to
encode a soluble protein of 249 amino acids. It shows significant similarities
to the ribosomal protein L10 from various bacteria and to a previously
determined amino-terminal peptide sequence of the yeast mitochondrial ribosomal
protein L11. The predicted amino-acid sequence of YDL202w starts with a stretch
which has neither any correspondence in the bacterial sequences nor in the
protein isolated from mitochondrial ribosomes. Furthermore, this stretch matches
the requirements for a signal sequence for mitochondrial protein import. A
mitochondrial location of the YDL202w gene product was proven by use of a
carboxy terminally HA-tagged version. These findings clearly indicate that
YDL202w encodes this mitochondrial ribosomal protein (YmL11).

PMID: 9162110 [PubMed - indexed for MEDLINE]



NR97: Microbiol Mol Biol Rev. 1997 Mar;61(1):90-104. 

Evolutionary divergence and salinity-mediated selection in halophilic archaea.

Dennis PP, Shimmin LC.

Department of Biochemistry & Molecular Biology, University of British Columbia,
Vancouver, Canada. patrick.p.dennis@unixg.ubc.ca

Halophilic (literally salt-loving) archaea are a highly evolved group of
organisms that are uniquely able to survive in and exploit hypersaline
environments. In this review, we examine the potential interplay between
fluctuations in environmental salinity and the primary sequence and tertiary
structure of halophilic proteins. The proteins of halophilic archaea are highly
adapted and magnificently engineered to function in an intracellular milieu that
is in ionic balance with an external environment containing between 2 and 5 M
inorganic salt. To understand the nature of halophilic adaptation and to
visualize this interplay, the sequences of genes encoding the L11, L1, L10, and
L12 proteins of the large ribosome subunit and Mn/Fe superoxide dismutase
proteins from three genera of halophilic archaea have been aligned and analyzed
for the presence of synonymous and nonsynonymous nucleotide substitutions.
Compared to homologous eubacterial genes, these halophilic genes exhibit an
inordinately high proportion of nonsynonymous nucleotide substitutions that
result in amino acid replacement in the encoded proteins. More than one-third of
the replacements involve acidic amino acid residues. We suggest that
fluctuations in environmental salinity provide the driving force for fixation of
the excessive number of nonsynonymous substitutions. Tinkering with the number,
location, and arrangement of acidic and other amino acid residues influences the
fitness (i.e., hydrophobicity, surface hydration, and structural stability) of
the halophilic protein. Tinkering is also evident at halophilic protein
positions monomorphic or polymorphic for serine; more than one-third of these
positions use both the TCN and the AGY serine codons, indicating that there have
been multiple nonsynonymous substitutions at these positions. Our model suggests
that fluctuating environmental salinity prevents optimization of fitness for
many halophilic proteins and helps to explain the unusual evolutionary
divergence of their encoding genes.

Publication Types:
    Review

PMID: 9106366 [PubMed - indexed for MEDLINE]



NR98: Biochimie. 1997;79(1):7-11. 

Molecular characterization of the prokaryotic efp gene product involved in a
peptidyltransferase reaction.

Aoki H, Adams SL, Turner MA, Ganoza MC.

Banting and Best Department of Medical Research, University of Toronto, Ontario,
Canada.

The translation factor EF-P is required for efficient prokaryotic peptide bond
synthesis on 70S ribosomes from fMet-tRNAfMet. This protein has been purified
from Escherichia coli cells and the gene, efp, encoding it has been cloned and
sequenced. We have isolated recombinant clones which overexpress a protein that
co-migrates with purified EF-P upon SDS-PAGE analysis. Using these clones, we
report the purification, crystallization and initial characterization of the efp
gene product. The mechanism by which EF-P stimulates peptide-bond synthesis was
studied using several antibiotics that inhibit translocation, peptide-bond
synthesis and decoding. The stimulation of peptidyltransferase by EF-P was not
inhibited by antibiotics that affect translocation and occupation of the A site
(in the elongation state), ie thiostrepton, viomycin, neomycin and fusidic acid
but was inhibited by streptomycin as well as by inhibitors of
peptidyltransferase, chloramphenicol and lincomycin. This observation and the
requirement for L16 but not for the L7/L12 nor L6 or L11 r-proteins suggest that
the binding site for EF-P may overlap the peptidyltransferase center of the
ribosome.

PMID: 9195040 [PubMed - indexed for MEDLINE]



NR99: Nat Struct Biol. 1997 Jan;4(1):70-7. 

High resolution solution structure of ribosomal protein L11-C76, a helical
protein with a flexible loop that becomes structured upon binding to RNA.

Markus MA, Hinck AP, Huang S, Draper DE, Torchia DA.

Molecular Structural Biology Unit, National Institute of Dental Research,
Bethesda, Maryland 20892-4320, USA.

The structure of the C-terminal RNA recognition domain of ribosomal protein L11
has been solved by heteronuclear three-dimensional nuclear magnetic resonance
spectroscopy. Although the structure can be considered high resolution in the
core, 15 residues between helix alpha 1 and strand beta 1 form an extended,
unstructured loop. 15N transverse relaxation measurements suggest that the loop
is moving on a picosecond-to-nanosecond time scale in the free protein but not
in the protein bound to RNA. Chemical shifts differences between the free
protein and the bound protein suggest that the loop as well as the C-terminal
end of helix alpha 3 are involved in RNA binding.

PMID: 8989327 [PubMed - indexed for MEDLINE]



NR100: Nat Struct Biol. 1997 Jan;4(1):24-7. 

The RNA binding domain of ribosomal protein L11 is structurally similar to
homeodomains.

Xing Y, Guha Thakurta D, Draper DE.

The RNA binding domain of ribosomal protein L11 is strikingly similar to the
homeodomain class of eukaryotic DNA binding proteins: it contains three
alpha-helices that superimpose with homeodomain alpha-helices, and some
conserved residues required for rRNA recognition align with homeodomain helix
III residues contacting DNA bases.

Publication Types:
    Letter

PMID: 8989317 [PubMed - indexed for MEDLINE]



NR101: Nucleic Acids Res. 1996 Jul 15;24(14):2666-72. 

Structure of a U.U pair within a conserved ribosomal RNA hairpin.

Wang YX, Huang S, Draper DE.

Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.

A conserved hairpin corresponding to nt 1057-1081 of large subunit rRNA
(Escherichia coli numbering) is part of a domain targeted by antibiotics and
ribosomal protein L11. The stem of the hairpin contains a U.U juxtaposition,
found as either U.U or U.C in virtually all rRNA sequences. This hairpin has
been synthesized and most of the aromatic and sugar protons were assigned by
two-dimensional proton NMR. Distances and sugar puckers deduced from the NMR
data were combined with restrained molecular dynamics calculations to deduce
structural features of the hairpin. The two U residues are stacked in the helix,
form one NH3-O4 hydrogen bond and require an extended backbone conformation
(trans alpha and gamma) at one of the U nucleotides. The hairpin loop, UAGAAGC
closed by a U-A pair, is the same size as tRNA anticodon loops, but not as well
ordered.

PMID: 8758993 [PubMed - indexed for MEDLINE]



NR102: Gene. 1996 May 24;171(1):95-8. 

The unique organization of the rpoB region of Spiroplasma citri: a restriction
and modification system gene is adjacent to rpoB.

Laigret F, Gaurivaud P, Bove JM.

Laboratoire de Biologie Cellulaire et Moleculaire, Institut National de la
Recherche Agronomique et Universite de Bordeaux II, Villenave d'Ornon, France.
laigret@bordeaux.inra.fr

A 6.5-kb DNA fragment containing the gene (rpoB) encoding the RNA polymerase
(RNAP) beta subunit, from the mollicute Spiroplasma citri (Sc), was cloned and
sequenced. The classical eubacterial organization, with the genes (rplK, A, J
and L) encoding ribosomal proteins L11, L1, L10 and L12 located immediately
upstream from rpoB, was not found in the Sc DNA. Instead, an open reading frame
(hsdS) potentially encoding a component of a type I restriction and modification
system was identified upstream from rpoB, and sequences showing similarities
with insertion elements were found between hsdS and rpoB.

PMID: 8675039 [PubMed - indexed for MEDLINE]



NR103: Gene. 1996 May 24;171(1):135-6. 

Gene organization in the ada-rplL region of Streptomyces virginiae.

Katayama M, Sakai Y, Okamoto S, Ihara F, Nihira T, Yamada Y.

Department of Biotechnology, Faculty of Engineering, Osaka University, Japan.

The gene organization of a 7.4-kb region of the Streptomyces virginiae (Sv)
chromosome was determined. The predicted open reading frames (ORFs) and their
predicted products, in sequence order, were (i) ada, encoding adenosine
deaminase [EC 3.5.4.4], (ii) aat, encoding a protein homologous to aspartate
aminotransferase [EC 2.6.1.1], (iii) secE, encoding a protein involved in
protein secretion, (iv) vbrA, encoding a NusG-like protein involved in
antitermination of transcription as described by Okamoto et al. [J. Biol. Chem.
267 (1992) 1093-1098], and (v) rplKAJL, encoding the large subunits of the
ribosomal proteins L11, L1, L10 and L12. Six of the ORFs (secE-rplL) were
oriented in the same direction, but the other two (ada and aat) had the opposite
orientation. The gene organization of the secE-rplL region in Sv was identical
to that in Escherichia coli.

PMID: 8675024 [PubMed - indexed for MEDLINE]



NR104: Biochemistry. 1996 Feb 6;35(5):1581-8. 

Cooperative interactions of RNA and thiostrepton antibiotic with two domains of
ribosomal protein L11.

Xing Y, Draper DE.

Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218,
USA.

Ribosomal protein L11 interacts with a 58-nucleotide domain of large subunit
ribosomal RNA; both the protein and its RNA target have been highly conserved.
The antibiotic thiostrepton recognizes the same RNA domain, and binds to the
ribosome cooperatively with L11. Experiments presented here show that RNA
recognition and thiostrepton cooperativity can be attributed to C- and
N-terminal domains of L11, respectively. Under trypsin digestion conditions that
degrade Bacillus stearothermophilus L11 to small fragments, the target RNA
protects the C-terminal 77 residues from digestion, and thiostrepton and RNA in
combination protect the entire protein. A 76-residue C-terminal fragment of L11
was overexpressed and shown to fold into a stable structure binding ribosomal
RNA with essentially the same properties as full-length L11. An
L11.thiostrepton.RNA complex was 100-200-fold more stable than expected on the
basis of L11-RNA and thiostrepton-RNA binding affinities; similar measurements
with the C-terminal fragment detected no cooperativity with thiostrepton. L11
function is thus more complex than simple interaction with ribosomal RNA; we
suggest that thiostrepton mimics some ribosomal component or factor that
normally interacts with the L11 N-terminal domain.

PMID: 8634289 [PubMed - indexed for MEDLINE]



NR105: Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12309-13. 

UGA suppression by a mutant RNA of the large ribosomal subunit.

Jemiolo DK, Pagel FT, Murgola EJ.

Department of Molecular Genetics, University of Texas M.D. Anderson Cancer
Center, Houston 77030, USA.

A role for rRNA in peptide chain termination was indicated several years ago by
isolation of a 168 rRNA (small subunit) mutant of Escherichia coli that
suppressed UGA mutations. In this paper, we describe another interesting rRNA
mutant, selected as a translational suppressor of the chain-terminating mutant
trpA (UGA211) of E. coli. The finding that it suppresses UGA at two positions in
trpA and does not suppress the other two termination codons, UAA and UAG, at the
same codon positions (or several missense mutations, including UGG, available at
one of the two positions) suggests a defect in UGA-specific termination. The
suppressor mutation was mapped by plasmid fragment exchanges and in vivo
suppression to domain II of the 23S rRNA gene of the rrnB operon. Sequence
analysis revealed a single base change of G to A at residue 1093, an almost
universally conserved base in a highly conserved region known to have specific
interactions with ribosomal proteins, elongation factor G, tRNA in the A-site,
and the peptidyltransferase region of 23S rRNA. Several avenues of action of the
suppressor mutation are suggested, including altered interactions with release
factors, ribosomal protein L11, or 16S rRNA. Regardless of the mechanism, the
results indicate that a particular residue in 23S rRNA affects peptide chain
termination, specifically in decoding of the UGA termination codon.

PMID: 8618891 [PubMed - indexed for MEDLINE]



NR106: J Biol Chem. 1995 Dec 15;270(50):29889-93. 

A base substitution within the GTPase-associated domain of mammalian 28 S
ribosomal RNA causes high thiostrepton accessibility.

Uchiumi T, Wada A, Kominami R.

Department of Biochemistry, Niigata University School of Medicine, Japan.

A molecular basis for the insensitivity of eukaryotic ribosomes to the
antibiotic thiostrepton was investigated using synthetic 100-nucleotide-long
fragments covering the GTPase domain of 23/28 S rRNA. Filter binding assay
showed no detectable binding of the rat RNA to thiostrepton, but the binding
capacity was markedly increased by base substitution of G1878 to A at the
position corresponding to 1067 of Escherichia coli 23 S rRNA. The association
constant (K alpha) for the rat A 1878 mutant was 0.60 x 10(6) M-1, which was
comparable with that of the E. coli RNA (K alpha = 1.1 x 10(6) M-1). This
suggests that the eukaryotic G 1878 participates in the resistance for
thiostrepton. On the other hand, the RNA fragments of the two species had a
similar binding capacity for E. coli ribosomal protein L11 and its mammalian
homologue L12. Gel electrophoresis under a high ionic condition, however,
revealed a difference between the two proteins. E. coli L11 formed stable
complexes with both the E. coli RNA and the rat A 1878 mutant RNA in the
presence of thiostrepton, while rat L12 failed to exhibit such complex
formation. This suggests that the eukaryotic L12 protein may also be an element
giving the resistance for thiostrepton. These results are discussed in terms of
preserved three-dimensional conformation of the RNA backbone between prokaryotes
and higher eukaryotes.

PMID: 8530386 [PubMed - indexed for MEDLINE]



NR107: Biochem Cell Biol. 1995 Nov-Dec;73(11-12):1179-85. 

Recognition determinants for proteins and antibiotics within 23S rRNA.

Douthwalte S, Voldborg B, Hansen LH, Rosendahl G, Vester B.

Department of Molecular Biology, Odense University, Denmark.

Ribosomal RNAs fold into phylogenetically conserved secondary and tertiary
structures that determine their function in protein synthesis. We have
investigated Escherichia coli 23S rRNA to identify structural elements that
interact with antibiotic and protein ligands. Using a combination of molecular
genetic and biochemical probing techniques, we have concentrated on regions of
the rRNA that are connected with specific functions. These are located in
different domains within the 23S rRNA and include the ribosomal
GTPase-associated center in domain II, which contains the binding sites for
r-proteins L10.(L12)4 and L11 and is inhibited by interaction with the
antibiotic thiostrepton. The peptidyltransferase center within domain V is
inhibited by macrolide, lincosamide, and streptogramin B antibiotics, which
interact with the rRNA around nucleotide A2058. Drug resistance is conferred by
mutations here and by modification of A2058 by ErmE methyltransferase. ErmE
recognizes a conserved motif displayed in the primary and secondary structure of
the peptidyl transferase loop. Within domain VI of rRNA, the alpha-sarcin
stem-loop is associated with elongation factor binding and is the target site
for ribotoxins including the N-glycosidase ribosome-inactivating proteins ricin
and pokeweed antiviral protein (PAP). The orientations of the 23S rRNA domains
are constrained by tetiary interactions, including a pseudoknot in domain II and
long-range base pairings in the center of the molecule that bring domains II and
V closer together. The phenotypic effects of mutations in these regions have
been investigated by expressing 23S rRNA from plasmids. Allele-specific priming
sites have been introduced close to these structures in the rRNA to enable us to
study the molecular events there.

Publication Types:
    Review
    Review, Tutorial

PMID: 8722035 [PubMed - indexed for MEDLINE]



NR108: Biochem Cell Biol. 1995 Nov-Dec;73(11-12):959-68. 

Proteins P1, P2, and P0, components of the eukaryotic ribosome stalk. New
structural and functional aspects.

Remacha M, Jimenez-Diaz A, Santos C, Briones E, Zambrano R, Rodriguez Gabriel
MA, Guarinos E, Ballesta JP.

Centro de Biologia Molecular, C.S.I.C. and U.A.M., Madrid, Spain.

The eukaryoic ribosomal stalk is thought to consist of the phosphoproteins P1
and P2, which form a complex with protein PO. This complex interacts at the
GTPase domain in the large subunit rRNA, overlapping the binding site of the
protein L11-like eukaryotic counterpart (Saccharomyces cerevisiae protein L15
and mammalian protein L12). An unusual pool of the dephosphorylated forms of
proteins P1 and P2 is detected in eukaryotic cytoplasm, and an exchange between
the proteins in the pool and on the ribosome takes place during translation.
Quadruply disrupted yeast strains, carrying four inactive acidic protein genes
and, therefore, containing ribosomes totally depleted of acidic proteins, are
viable but grow with a doubling time threefold higher than wild-type cells. The
in vitro translation systems derived from these stains are active but the
two-dimensional gel electrophoresis pattern of proteins expressed in vivo and in
vitro is partially different. These results indicate that the P1 and P2 proteins
are not essential for ribosome activity but are able to affect the translation
of some specific mRNAs. Protein PO is analogous to bacterial ribosomal protein
L10 but carries an additional carboxyl domain showing a high sequence homology
to the acidic proteins P1 and P2, including the terminal peptide DDDMGFGLFD.
Successive deletions of the PO carboxyl domain show that removal of the last 21
amino acids from the PO carboxyl domain only slightly affects the ribosome
activity in a wild-type genetic background; however, the same deletion is lethal
in a quadruple disruptant deprived of acidic P1/P2 proteins. Additional
deletions affect the interaction of PO with the P1 and P2 proteins and with the
rRNA. The experimental data available support the implication of the eukaryotic
stalk components in some regulatory process that modulates the ribosomal
activity.

Publication Types:
    Review
    Review, Tutorial

PMID: 8722011 [PubMed - indexed for MEDLINE]



NR109: Biochem Cell Biol. 1995 Nov-Dec;73(11-12):925-31. 

Variety of nonsense suppressor phenotypes associated with mutational changes at
conserved sites in Escherichia coli ribosomal RNA.

Murgola EJ, Pagel FT, Hijazi KA, Arkov AL, Xu W, Zhao SQ.

Department of Molecular Genetics, University of Texas M.D. Anderson Cancer
Center, Houston 77030, USA.

To screen for ribosomal RNA mutants defective in peptide chain termination, we
have been looking for rRNA mutants that exhibit different patterns of
suppression of nonsense mutations and that do not suppress missense mutations at
the same positions in the same reporter gene. The rRNA mutations were induced by
segment-directed randomly mutagenic PCR treatment of a cloned rrnB operon,
followed by subcloning of the mutagenesis products and transformation of strains
containing different nonsense mutations in the Escherichia coli trpA gene. To
date, we have repeatedly obtained only two small sets of mutations, one in the
3' domain of 16S rRNA, at five nucleotides out of the 610 mutagenized (two in
helix 34 and three in helix 44), and the other in 23S rRNA at only four
neighboring nucleotide positions (in a highly conserved hexanucleotide loop)
within the 1.4 kb mutagenized segment. There is variety, however, in the
suppression patterns of the mutants, ranging from suppression of UAG or UGA,
through suppression of UAG and UGA, but not UAA, to suppression of all three
termination codons. The two helices in 16S rRNA have previously been associated
both physically and functionally with the decoding center of the ribosome. The
23S region is part of the binding site for the large subunit protein L11 and the
antibiotic thiostrepton, both of which have been shown to affect peptide chain
termination. Finally, we have demonstrated that the 23S mutant A1093, which
suppresses trpA UGA mutations very efficiently, is lethal at temperatures above
36 degrees C (when highly expressed). This lethality is overcome by secondary
23S rRNA mutations in domain V. Our results suggest that specific regions of 16S
and 23S rRNA are involved in peptide chain termination, that the lethality of
A1093 is caused by high-level UGA suppression, and that intramolecular
interaction between domains II and V of 23S rRNA may play a role in peptide
chain termination at the UGA stop codon.

PMID: 8722008 [PubMed - indexed for MEDLINE]



NR110: Nucleic Acids Res. 1995 Sep 11;23(17):3426-33. 

On the role of rRNA tertiary structure in recognition of ribosomal protein L11
and thiostrepton.

Lu M, Draper DE.

Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.

Ribosomal protein L11 and an antibiotic, thiostrepton, bind to the same highly
conserved region of large subunit ribosomal RNA and stabilize a set of
NH4(+)-dependent tertiary interactions within the domain. In vitro selection
from partially randomized pools of RNA sequences has been used to ask what
aspects of RNA structure are recognized by the ligands. L11-selected RNAs showed
little sequence variation over the entire 70 nucleotide randomized region, while
thiostrepton required a slightly smaller 58 nucleotide domain. All the selected
mutations preserved or stabilized the known secondary and tertiary structure of
the RNA. L11-selected RNAs from a pool mutagenized only around a junction
structure yielded a very different consensus sequence, in which the RNA tertiary
structure was substantially destabilized and L11 binding was no longer dependent
on NH4+. We propose that L11 can bind the RNA in two different 'modes',
depending on the presence or absence of the NH4(+)-dependent tertiary structure,
while thiostrepton can only recognize the RNA tertiary structure. The different
RNA recognition mechanisms for the two ligands may be relevant to their
different effects on protein synthesis.

PMID: 7567452 [PubMed - indexed for MEDLINE]



NR111: Nucleic Acids Res. 1995 Jul 11;23(13):2396-403. 

Cooperative assembly of proteins in the ribosomal GTPase centre demonstrated by
their interactions with mutant 23S rRNAs.

Rosendahl G, Douthwaite S.

Department of Molecular Biology, Odense University, Denmark.

The ribosomal protein L11 binds to the region of 23S rRNA associated with the
GTPase-dependent steps of protein synthesis. Nucleotides 1054-1107 within this
region of the Escherichia coli 23S rRNA gene were mutagenized with bisulphite.
Twenty point mutations (G-->A and C-->T transitions) and numerous multiple
mutations were generated. Expression of mutant 23S rRNAs in vivo shows that all
the mutations detectably alter the phenotype, with effects ranging from a slight
growth rate reduction to lack of viability. Temperature sensitivity is conferred
by 1071G-->A and 1092C-->U substitutions. These effects are relieved by point
mutations at other sites, indicating functional interconnections within the
higher order structure of this 23S rRNA region. Several mutations prevent direct
binding of r-protein L11 to 23S rRNA in vitro. These mutations are mainly in a
short irregular stem (1087-1102) and within a hairpin loop (1068-1072), where
the protein probably makes nucleotide contacts. Some of these mutations also
interfere with binding of the r-protein complex L10.(L12)4 to an adjacent site
on the rRNA. When added together to rRNA, proteins L10.(L12)4 and L11 bind
cooperatively to overcome the effects of mutations at 1091 and 1099. The
proteins also stimulate each others binding to rRNA mutated at 1087 or 1092,
although in these cases binding remains clearly substoichiometric. Surprisingly,
none of the mutations prevents incorporation of L11 into ribosomes in vivo,
indicating that other, as yet unidentified, factors are involved in the
cooperative assembly process.

PMID: 7630717 [PubMed - indexed for MEDLINE]



NR112: DNA Res. 1995 Jun 30;2(3):129-32. 

Mitochondrial ribosomal protein L11 gene of Dictyostelium discoideum resides not
in the nuclear genome but in the mitochondrial genome.

Iwamoto M, Yanagisawa K, Tanaka Y.

Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan.

During the course of analysis of the mitochondrial genome of the cellular slime
mold, Dictyostelium discoideum, we found a gene (rpl11) for mitochondrial
ribosomal protein (RPL11), having 172 amino acid residues. Southern blot
analysis revealed that the gene resided in the mitochondrial DNA as a
single-copy but not in the nuclear DNA. From Northern blot experiments, one
major mRNA (about 27 kb) and two minor mRNAs (about 4 and 5 kb) for the gene
were detected in the mitochondria. This is the first report showing that the
active gene for RPL11 still resides in the mitochondrial genome and has not been
transferred to the nuclear genome in D. discoideum.

PMID: 8581739 [PubMed - indexed for MEDLINE]



NR113: J Mol Biol. 1995 Jun 2;249(2):319-31. 

Stabilization of a ribosomal RNA tertiary structure by ribosomal protein L11.

Xing Y, Draper DE.

Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.

Interactions between ribosomal protein L11 and a domain of large subunit rRNA
have been highly conserved and are essential for efficient protein synthesis. To
study the effects of L11 on rRNA folding, a homolog of the Escherichia coli L11
gene has been amplified from Bacillus stearothermophilus DNA and cloned into a
phage T7 polymerase-based expression system. The expressed protein is 93%
homologous to the L11 homolog from Bacillus subtilis, denatures at temperatures
above 72 degrees C, and has nearly identical rRNA binding properties as the
Escherichia coli L11 in terms of RNA affinity constants and their dependences on
temperature, Mg2+ concentration, monovalent cation, and RNA mutations. Mg2+ and
NH4+ are specifically bound by the RNA-protein complex, with apparent ion-RNA
affinities of 1.6 mM-1 and 19 M-1, respectively, at 0 degree C. The effect of
the thermostable L11 on the unfolding of a 60 nucleotide rRNA fragment
containing its binding domain has been examined in melting experiments. The
lowest temperature RNA transition, which is attributed to tertiary structure
unfolding, is stabilized by approximately 25 degrees C, and the interaction has
an intrinsic enthalpy of approximately 13 kcal/mol. The thermal stability of the
protein-RNA complex is enhanced by increasing Mg2+ concentration and by NH4+
relative to Na+. Thus L11, NH4+, and Mg2+ all bind and stabilize the same rRNA
tertiary interactions, which are conserved and presumably important for ribosome
function.

PMID: 7783196 [PubMed - indexed for MEDLINE]



NR114: Mol Biochem Parasitol. 1995 May;71(2):261-4. 

Cloning and characterization of the ribosomal l11 gene from Leishmania spp.

Love DC, Wilson ME, Mosser DM.

Department of Microbiology and Immunology, Temple University School of Medicine,
Philadelphia, PA 19140, USA.

PMID: 7477109 [PubMed - indexed for MEDLINE]



NR115: Mol Gen Genet. 1995 Apr 10;247(1):118-22. 

Cloning, nucleotide sequence, and transcriptional analysis of the nusG gene of
Streptomyces coelicolor A3(2), which encodes a putative transcriptional
antiterminator.

Puttikhunt C, Nihira T, Yamada Y.

Department of Biotechnology, Faculty of Engineering, Osaka University, Japan.

A 3 kb genomic fragment containing the nusG gene of Streptomyces coelicolor
A3(2) was identified, cloned and sequenced. Sequence analysis revealed 3
complete and 2 truncated open reading frames (ORFs): truncated ORFU (similar to
a Bacillus gene encoding a thermostable aspartate aminotransferase)-secE (94
amino acids; 79.0% similarity to Escherichia coli SecE)-nusG (300 amino acids;
73.3% similarity to E. coli NusG)-rplK (144 amino acids; 88.5% similarity to E.
coli ribosomal subunit L11)-truncated rplA (similar to E. coli ribosomal subunit
L1). The gene organization secE-nusG-rplKA exactly matches that in E. coli.
Transcriptional analyses by the primer extension method revealed one
transcriptional start site each for secE and nusG, and two sites for rplK. The
presence of promoters was also confirmed with the aid of a promoter-probe
vector.

PMID: 7715599 [PubMed - indexed for MEDLINE]



NR116: Biochim Biophys Acta. 1995 Mar 14;1261(1):147-50. 

Molecular cloning of the Drosophila homologue of the rat ribosomal protein L11
gene.

Larochelle S, Suter B.

Department of Biology, McGill University, Montreal, Canada.

We report the isolation of the Drosophila melanogaster homologue of the rat
ribosomal protein L11 gene. The gene is present in the Drosophila genome at
polytene chromosome location 56D, on the right arm of the second chromosome. The
Drosophila DL11 gene appears to encode two messages of 0.8 and 0.9 kb which are
expressed throughout development with variations in their relative abundance.
DL11 codes for a predicted protein of 184 amino acids with a molecular mass of
21.1 kDa.

PMID: 7893752 [PubMed - indexed for MEDLINE]



NR117: Bioorg Khim. 1995 Feb;21(2):158-60. 

[Cloning and determination of the primary structure of DNA complementary to the
mRNA of human ribosomal protein L11]

[Article in Russian]

Mishin VP, Filipenko ML, Muravlev AI, Karpova GG, Mertvetsov NP.

A polymerase chain reaction strategy was employed to isolate cDNA encoding L11
human ribosomal protein. Based on the known nucleotide sequence of 5'-region of
the ribosomal protein L11 mRNA, we have designed primers and used them in
amplification of corresponding sequence of human cDNA from total placenta cDNA.
The fragment of RPS26 cDNA was cloned in plasmid vector and sequenced. Sequence
analysis showed that there is high homology (88%) between coding regions of
RPS26 mRNAs in rat liver and human placenta. The amino acid exchanges were
observed at positions: 91 (Asp-->Glu), 217 (Thr-->Ala), 352 (Lys-->Glu).

Publication Types:
    Letter

PMID: 7748210 [PubMed - indexed for MEDLINE]



NR118: Nucleic Acids Symp Ser. 1995;(33):70-2. 

Mutations at three sites in the Escherichia coli 23S ribosomal RNA binding
region for protein L11 cause UGA-specific suppression and conditional lethality.

Murgola EJ, Xu W, Arkov AL.

Department of Molecular Genetics, University of Texas M. D. Anderson Cancer
Center, Houston 77030, USA.

A single nucleotide change, G to A, at nucleotide position 1093 of E. coli 23S
ribosomal RNA was found to cause UGA-specific suppression (D.K. Jemiolo, F.T.
Pagel and E.J. Murgola, Proc. Natl. Acad. Sci. USA, in press). To obtain new
kinds of UGA-specific suppressors in 23S rRNA, we used segment-directed
mutagenic PCR, and targeted first the 1405 nucleotide SnaBI/I-CeuI segment,
which includes position 1093, of the rrnB operon cloned into a multicopy
plasmid. The mutagenized fragments were subcloned into the plasmid vector and
used to transform to ampicillin resistance (Ampr) a recipient strain containing
a UGA mutation in trpA. The Ampr transformants were then screened for
suppression of UGA. After purification, Trp+ transformants were tested for
association of the suppressor phenotype first with the plasmid and then
specifically with the SnaBI/I-CeuI fragment. In one screening, four different
kinds of mutational change were found, all at three sites within a highly
conserved hexanucleotide loop in domain II of 23S rRNA. This region is part of
the site for binding of the large subunit protein L11, which has been shown to
be involved in peptide chain termination in a specific way. All of the mutants
(G1093A, G1093 delta, A1095 delta, and U1097 delta) suppress UGA mutations, but
not UAA or UAG mutations, and all four types exhibit high-temperature
conditional lethality when highly expressed. Several mechanisms can be suggested
for the UGA-specific suppression exhibited by these mutants, including altered
interaction with protein L11, Second-site mutations that overcome the
conditional lethality of G1093A indicate that intramolecular interactions within
23S rRNA may play a role in peptide chain termination at the UGA stop codon.

PMID: 8643403 [PubMed - indexed for MEDLINE]



NR119: Nucleic Acids Symp Ser. 1995;(33):5-7. 

Protein recognition of a ribosomal RNA tertiary structure.

Draper DE, Xing Y.

Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.

Ribosomal protein L11 recognizes a highly conserved, 58 nucleotide domain of
large subunit ribosomal RNA. This domain has a set of tertiary interactions that
are specifically stabilized by Mg2+ and NH4+ ions. The protein recognizes this
tertiary structure, in the sense that ions stabilizing the tertiary structure
also promote L11 binding, and a heat stable form of the protein (from Bacillus
stearothermophilus) prevents RNA unfolding. We also find that these RNA-binding
properties are confined to a approximately 75 amino acid domain of the protein.
The larger issue of L11 function within the ribosome is discussed in light of
these findings.

Publication Types:
    Review
    Review, Tutorial

PMID: 8643396 [PubMed - indexed for MEDLINE]



NR120: Mol Microbiol. 1994 Dec;14(5):947-58. 

Ribosomal protein methylation in Escherichia coli: the gene prmA, encoding the
ribosomal protein L11 methyltransferase, is dispensable.

Vanet A, Plumbridge JA, Guerin MF, Alix JH.

Institut de Biologie Physico-Chimique, URA1139 CNRS, Paris, France.

The prmA gene, located at 72 min on the Escherichia coli chromosome, is the
genetic determinant of ribosomal protein L11-methyltransferase activity.
Mutations at this locus, prmA1 and prmA3, result in a severely undermethylated
form of L11. No effect, other than the lack of methyl groups on L11, has been
ascribed to these mutations. DNA sequence analysis of the mutant alleles prmA1
and prmA3 detected point mutations near the C-terminus of the protein and
plasmids overproducing the wild-type and the two mutant proteins have been
constructed. The wild-type PrmA protein could be crosslinked to its
radiolabelled substrate, S-adenosyl-L-methionine (SAM), by u.v. irradiation
indicating that it is the gene for the methyltransferase rather than a
regulatory protein. One of the mutant proteins, PrmA3, was also weakly
crosslinked to SAM. Both mutant enzymes when expressed from the overproducing
plasmids were capable of catalysing the incorporation of 3H-labelled methyl
groups from SAM to L11 in vitro. This confirmed the observation that the mutant
proteins possess significant residual activity which could account for their
lack of growth phenotype. However, a strain carrying an in vitro-constructed
null mutation of the prmA gene, transferred to the E. coli chromosome by
homologous recombination, was perfectly viable.

PMID: 7715456 [PubMed - indexed for MEDLINE]



NR121: J Mol Biol. 1994 Nov 25;244(2):242-9. 

Structure and transcription of the L11-L1-L10-L12 ribosomal protein gene operon
from the extreme thermophilic archaeon Sulfolobus acidocaldarius.

Ramirez C, Shimmin LC, Leggatt P, Matheson AT.

Department of Biochemistry and Microbiology, University of Victoria, B.C.,
Canada.

We have cloned and sequenced four ribosomal protein genes from the extreme
thermophilic archaeon Sulfolobus acidocaldarius P1. These genes code for
proteins equivalent to L11, L1, L10 and L12 from Escherichia coli. The genes for
the Sulfolobus L11, L1, L10 and L12 proteins are arranged in the same order as
the equivalent genes in E. coli, i.e. L11-L1-L10-L12, and are transcribed as a
single unit. Sequences resembling the consensus sequence for archaeal promoters
have been detected upstream of the transcription initiation site. Transcription
ends at several sites following a pyrimidine-rich region. The genes for proteins
L11, L10 and L1 start with unusual initiation codons: GUG in the case of the L1
and L10 genes; and UUG in the case of L11. There are overlapping stop/start
codons between the L11 and L1 genes, and between the L1 and L10, suggesting that
the translation of the four genes might be coupled as in the bacteria.

PMID: 7966335 [PubMed - indexed for MEDLINE]



NR122: Eur J Biochem. 1994 Sep 1;224(2):431-7. 

Overexpression of the thiostrepton-resistance gene from Streptomyces azureus in
Escherichia coli and characterization of recognition sites of the 23S rRNA A1067
2'-methyltransferase in the guanosine triphosphatase center of 23S ribosomal
RNA.

Bechthold A, Floss HG.

Department of Chemistry, University of Washington, Seattle 98195.

The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase
from Streptomyces azureus has been overexpressed in Escherichia coli using a
T7-RNA-polymerase-dependent expression vector. The protein was efficiently
expressed at levels up to 20% of total soluble protein and purified to near
homogeneity. Kinetic parameters for S-adenosyl-L-methionine (Km = 0.1 mM) and an
RNA fragment containing nucleotides 1029-1122 of the 23S ribosomal RNA from E.
coli (Km = 0.001 mM) were determined. S-Adenosyl-L-homocysteine showed
competitive product inhibition (Ki = 0.013 mM). Binding of either thiostrepton
or protein L11 inhibited methylation. RNA sequence variants of the RNA fragment
with mutations in nucleotides 1051-1108 were tested as substrates for the
methylase. The experimental data indicate that methylation is dependent on the
secondary structure of the hairpin including nucleotide A1067 and the exact
sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand.

PMID: 7925357 [PubMed - indexed for MEDLINE]



NR123: Mol Microbiol. 1994 May;12(3):375-85. 

Synthesis of ribosomal proteins during growth of Streptomyces coelicolor.

Blanco G, Rodicio MR, Puglia AM, Mendez C, Thompson CJ, Salas JA.

Departamento de Biologia Funcional, Universidad de Oviedo, Spain.

Changes in expression of ribosomal protein genes during growth and stationary
phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins
being synthesized were pulse-labelled with [35S]-methionine, separated by
two-dimensional polyacrylamide gel electrophoresis, and quantified using the
BioImage computer software. Most of the ribosomal proteins were synthesized
throughout the life cycle. Exceptions were two proteins whose synthesis
drastically decreased at the approach of stationary phase. These two proteins
were identified in purified ribosomes as homologues of Escherichia coli
ribosomal proteins L10 and L7/L12, using antibodies raised against fusion
proteins between these ribosomal proteins and Escherichia coli
beta-galactosidase. The genes (rplJ and rplL) encoding the L10 and L7/L12
proteins were contained in a 1.2 kb BamHI fragment that was cloned and
sequenced. The linkage and order of the genes coincide with other L10-L7/L12
operons. However, L11 and L1 genes were not present immediately upstream of the
L10 gene, as is the case for E. coli and other bacteria. Instead, two open
reading frames of unknown function were found immediately upstream of the L10
gene, in an adjacent 1.9 kb BamHI fragment.

PMID: 7545948 [PubMed - indexed for MEDLINE]



NR124: J Mol Evol. 1994 Apr;38(4):405-19. 

Molecular phylogenies based on ribosomal protein L11, L1, L10, and L12
sequences.

Liao D, Dennis PP.

Canadian Institute for Advanced Research, University of British Columbia,
Vancouver.

Available sequences that correspond to the E. coli ribosomal proteins L11, L1,
L10, and L12 from eubacteria, archaebacteria, and eukaryotes have been aligned.
The alignments were analyzed qualitatively for shared structural features and
for conservation of deletions or insertions. The alignments were further
subjected to quantitative phylogenetic analysis, and the amino acid identity
between selected pairs of sequences was calculated. In general, eubacteria,
archaebacteria, and eukaryotes each form coherent and well-resolved
nonoverlapping phylogenetic domains. The degree of diversity of the four
proteins between the three groups is not uniform. For L11, the eubacterial and
archaebacterial proteins are very similar whereas the eukaryotic L11 is clearly
less similar. In contrast, in the case of the L12 proteins and to a lesser
extent the L10 proteins, the archaebacterial and eukaryotic proteins are similar
whereas the eubacterial proteins are different. The eukaryotic L1 equivalent
protein has yet to be identified. If the root of the universal tree is near or
within the eubacterial domain, our ribosomal protein-based phylogenies indicate
that archaebacteria are monophyletic. The eukaryotic lineage appears to
originate either near or within the archaebacterial domain.

PMID: 8007008 [PubMed - indexed for MEDLINE]



NR125: J Mol Biol. 1994 Jan 28;235(4):1251-60. 

Location of the streptomycin ribosomal binding site explains its pleiotropic
effects on protein biosynthesis.

Abad JP, Amils R.

Centro de Biologia Molecular Severo Ochoa Facultad de Ciencias, Universidad
Autonoma de Madrid, Cantoblanco, Spain.

Photoaffinity-labeling experiments using three nitroguaiacol ether streptomycin
derivatives with spacers of different lengths between the antibiotic and the
photoreactive moiety (8, 12 and 17 A) allow us to: (1) unambiguously locate the
boundaries of the antibiotic binding site; and (2) test the topographical
consistency of the photolabeling results. The streptomycin binding site is
located in the interface between the ribosomal subunits, close to proteins S5 in
the 30 S and to L11 in the 50 S ribosomal subunits. This location explains most
of the antibiotic's pleiotropic effects on protein biosynthesis, especially
those related to the tRNA selection mechanism, and it also correlates with the
location of the ribosomal components involved in the different streptomycin
phenotypes.

PMID: 7508514 [PubMed - indexed for MEDLINE]



NR126: J Bacteriol. 1994 Jan;176(2):409-18. 

Autogenous translational regulation of the ribosomal MvaL1 operon in the
archaebacterium Methanococcus vannielii.

Hanner M, Mayer C, Kohrer C, Golderer G, Grobner P, Piendl W.

Institute of Medical Microbiology, University of Innsbruck, Austria.

The mechanisms for regulation of ribosomal gene expression have been
characterized in eukaryotes and eubacteria, but not yet in archaebacteria. We
have studied the regulation of the synthesis of ribosomal proteins MvaL1,
MvaL10, and MvaL12, encoded by the MvaL1 operon of Methanococcus vannielii, a
methanogenic archaebacterium. MvaL1, the homolog of the regulatory protein L1
encoded by the L11 operon of Escherichia coli, was shown to be an autoregulator
of the MvaL1 operon. As in E. coli, regulation takes place at the level of
translation. The target site for repression by MvaL1 was localized by
site-directed mutagenesis to a region within the coding sequence of the MvaL1
gene commencing about 30 bases downstream of the ATG initiation codon. The MvaL1
binding site on the mRNA exhibits similarity in both primary sequence and
secondary structure to the L1 regulatory target site of E. coli and to the
putative binding site for MvaL1 on the 23S rRNA. In contrast to other regulatory
systems, the putative MvaL1 binding site is located in a sequence of the mRNA
which is not in direct contact with the ribosome as part of the initiation
complex. Furthermore, the untranslated leader sequence is not involved in the
regulation. Therefore, we suggest that a novel mechanism of translational
feedback regulation exists in M. vannielii.

PMID: 8288536 [PubMed - indexed for MEDLINE]



NR127: Mol Biol (Mosk). 1994 Jan-Feb;28(1):82-6. 

[Study of segments of 23S RNA, important for interaction with elongation factors
Tu and G using complementary DNA-oligonucleotides]

[Article in Russian]

Bubunenko MG, Gudkov AT.

Several oligonucleotides complementary to different 23S RNA regions were tested
in the elongation factor-dependent reactions of the ribosomes. It was found that
the 1088-1100 and 1127-1140 sequence parts of the 23S RNA (binding regions for
the L11 protein) are very important for EF-G function. The EF-Tu function is
markedly less affected by these nucleotides. The probable role of 23S RNA
function is discussed.

PMID: 8145758 [PubMed - indexed for MEDLINE]