From the Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101
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ABSTRACT |
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The methyltransferase that forms
m2G1207 in Escherichia coli small subunit
rRNA has been purified, cloned, and characterized. The gene was
identified from the N-terminal sequence of the purified enzyme as the
open reading frame yjjT (SWISS-PROT accession number P39406). The gene, here renamed rsmC in view of its newly established function, codes for a 343-amino acid protein that has
homologs in prokaryotes, Archaea, and possibly also in lower eukaryotes. The enzyme reacted well with 30 S subunits reconstituted from 16 S RNA transcripts and 30 S proteins but was almost inactive with the corresponding free RNA. By hybridization and protection of
appropriate segments of 16 S RNA that had been extracted from 30 S
subunits methylated by the enzyme, it was shown that of the three
naturally occurring m2G residues, only m2G1207
was formed. Whereas close to unit stoichiometry of methylation could be
achieved at 0.9 mM Mg2+, both 2 mM
EDTA and 6 mM Mg2+ markedly reduced the level
of methylation, suggesting that the optimal substrate may be a
ribonucleoprotein particle less structured than a 30 S ribosome but
more so than free RNA.
The modified nucleosides of RNA remain one of the enigmas of RNA
biology. Despite being widely distributed among all classes of RNA in a
bewildering plethora of shapes and forms (1), little or no functional
role, and certainly no unifying concept, has yet to emerge. Although
certain modulating effects on cellular processes have been described,
usually under special circumstances and so far only in tRNA (2), no
modified nucleoside has yet been shown to be of major importance for
cell growth and/or survival. In rRNA, which next to tRNA contains the
widest known assortment of modified nucleosides, no function has so far
been ascribed to them, although a potential role in peptide bond
formation for a subset of the modified nucleosides of Escherichia
coli 23 S RNA has been postulated (3). In E. coli 16 S
RNA, in which all of the modified nucleosides are known and their
locations precisely determined, none was essential for any of the known ribosomal functions, although ribosomal efficiency was reduced to
approximately half when all were absent (4, 5). Evidence has been
presented that this lowered efficiency of the 30 S subunits may be
attributable to the lack of one or more of the 10 methylated residues
and/or the single pseudouridine (6) in the RNA (7).
To explore the role of individual modified nucleosides of 16 S RNA in
30 S subunit structure and function, a way is needed to specifically
block their formation, one at a time. The most straightforward way to
do this is by inactivating the enzymes responsible for their synthesis
by gene deletion or disruption, because mutation of the parent
nucleoside in the RNA could have unforeseen consequences. So far, three
genes and enzymes for the modified nucleosides of E. coli 16 S RNA have been described. The first, rsmA (more commonly
known as ksgA), makes the two m26A
residues at positions 1518 and 1519 (8). Loss of these four methyl
groups has little effect on ribosome function (9). The synthase for the
single pseudouridine at position 516 (6) has been
deleted,1 and the cell still
grows well, although precise growth rates have not yet been determined.
A methyltransferase that specifically forms m5C967 has
recently been described,2 but
results of deletion experiments are not yet available. In this
work, we describe the characterization of another
methyltransferase, that for m2G1207. The gene was
identified by N-terminal amino acid sequence analysis of the isolated
enzyme and confirmed by overexpression with a His tag, affinity
purification, and in vitro biochemical characterization.
Materials--
[3H]S-Adenosyl-methionine
([3H]-SAM)3 was
from Amersham Pharmacia Biotech. Plasmid pET-15b, the BL21/DE3 strain
of E. coli, and His-Bind resin were from Novagen, Inc. The
XL1-Blue Epicurian strain of E. coli was from Stratagene.
RNase T1 was from Calbiochem, and RNase P1 was obtained from Life
Technologies, Inc. DNase I was from Worthington. T4 DNA ligase and
restriction enzymes were from New England Biolabs. Wizard DNA
purification kits and RNasin were from Promega. BA85 cellulose nitrate
filters were obtained from Schleicher & Schuell, polyvinylidene
difluoride membranes were from Millipore, and omega cells were from
Filtron. DEAE-Sepharose CL-6B and MonoS fast protein liquid
chromatography columns were obtained from Amersham Pharmacia Biotech.
Alumina type A-5 was from Sigma. Deoxyoligonucleotides for protection
studies were those described previously (11). Polymerase chain reaction
primers were prepared as described (12).
Buffers--
Buffer Ax is 20 mM Hepes,
pH 7.5, 10 mM Mg(OAc)2, x mM
NH4Cl, and 2 mM dithiothreitol. Buffer
Bx is 20 mM Hepes, pH 7.5, 1 mM
EDTA, x mM NH4Cl, and 2 mM
dithiothreitol. Buffer C is 20 mM Hepes, pH 7.8, 20 mM NH4Cl, 5 mM mercaptoethanol
(BME), 0.1 mM EDTA, and 10% glycerol. Buffer D is 20 mM Hepes, pH 8.0, 100 mM NH4Cl, 5 mM BME, 0.1 mM EDTA, and 6 M urea.
Buffer LB is 50 mM Tris, pH 6.8, 100 mM
dithiothreitol, 2% SDS, 10% glycerol, and 0.1% bromphenol blue.
Binding Buffer is 20 mM Hepes, pH 7.9, 0.5 M
NaCl, and 5 mM imidazole. Elution Buffer is Binding Buffer
with the imidazole concentration raised to 1.0 M.
Purification of the m2G Methyltransferase--
The
purification procedure was similar to that previously described (13,
14). One hundred grams of E. coli MRE600 cells harvested in
mid-log phase were ground with 200 g of alumina. Then 3000 units
of DNase I and 100 ml of Buffer A100 containing 0.2 mM phenylmethanesulfonyl fluoride were added. The mixture was incubated for 15 min at 0 °C and then centrifuged in a Sorvall SS-34 rotor for 30 min at 16,000 rpm yielding the S30. The S30 supernatant was centrifuged for 4 h at 30,000 rpm in a Beckman Ti45 rotor. The pellet was resuspended in 80 ml of Buffer
A1000, underlayered with 40 ml of the same buffer plus 37%
sucrose, and centrifuged at 45,000 rpm for 16-18 h using slow
acceleration. The supernatant of this centrifugation is the high salt
wash. The high salt wash was adjusted to 35% saturation
(NH4)2SO4 and allowed to stir on
ice for 4 h. The precipitate was removed by centrifugation, and
the supernatant was dialyzed overnight against Buffer B50.
The dialyzed sample was loaded onto a 5 × 43-cm DEAE-Sepharose CL-6B column equilibrated with Buffer B50 and eluted with a
linear gradient of 50-500 mM NH4Cl in Buffer
B0. The m2G methyltransferase activity eluted
with a peak at 170 mM NH4Cl. The peak fractions
were pooled, concentrated, and returned to Buffer B50 by
omega cell filtration. The sample was loaded onto a 0.5 × 5-cm
MonoS column, which had been equilibrated with Buffer B50.
The column was eluted with a linear gradient from 50-1000 mM NH4Cl in Buffer B0. The
methyltransferase activity eluted at 250 mM
NH4Cl. The peak fractions were pooled and either used
directly or made 50% in glycerol for storage at Cloning and Overexpression of the m2G
Methyltransferase Gene--
The putative gene was amplified by
polymerase chain reaction. The N-terminal primer extended from
For overexpression the transformed BL21/DE3 cells were grown in
Luria-Bertani broth containing 50 µg/ml carbenicillin at 37 °C to
an A600 of 0.7. Isopropyl- Affinity Purification--
The S15 supernatant was dialyzed
against Binding Buffer immediately before application to a 2.5-ml
column of His-Bind resin. When Binding Buffer was used in the
sonication step, dialysis could be omitted. Conditions of preparation
and operation of the His-Bind resin column were as described in the
pET System Manual, 4th Ed. (Novagen). After addition of
Elution Buffer, the tagged protein was released. The
A280-containing fractions were pooled and dialyzed against
Buffer C. The S15 pellet from the same culture was solubilized in
Buffer D or Binding Buffer containing 6 M urea. When the
pellet was solubilized in Buffer D, the solution was dialyzed
versus Binding Buffer containing 6 M urea before
being applied to a 2.5-ml His-Bind column equilibrated in the same
buffer. Otherwise, it was applied directly after solubilization.
Elution was with Elution Buffer plus 6 M urea. The pooled
A280-containing fractions were dialyzed against Buffer D
with decreasing concentrations of urea from 6 to 3 M at 1.0 M intervals and then from 3 to 0 M at 0.5 M intervals for 1 h each. Both protein solutions were adjusted to contain 50% glycerol and stored at Methylase Assays--
Reaction mixtures contained 100 mM Hepes, pH 7.5, and 2 mM Mg(OAc)2
except where otherwise indicated, 200 mM NH4Cl,
5 mM dithiothreitol or BME, 2 µM
[3H]SAM, 500 units/ml RNasin, and 100 nM 16 S
RNA transcript (prepared as in Ref. 15) or 80 nM 30 S
ribosomal subunits reconstituted from 16 S RNA transcripts and
ribosomal proteins (4, 16), and enzyme as indicated. Incubation was at
37 °C. The reaction was stopped by the addition of ice-cold 5%
trichloroacetic acid. After precipitation at 0 °C for 10 min,
samples were collected on BA85 cellulose nitrate filters, the filters
were dissolved in scintillation fluid, and the radioactivity was
measured. One unit of activity equals 1 pmol of methyl incorporated in
30 min under the above conditions.
Nucleoside Analysis--
Recombinant m2G
methyltransferase was used to 3H methylate 30 S particles
as described above. 1.05 pmol of CH3/pmol of particles was obtained.
After phenol extraction of the [3H]RNA, it was digested
in a 250-µl incubation mixture containing 20 mM NaOAc,
0.5 mM ZnSO4, 40 pmol/ml carrier 16 S RNA, 80 µg/ml RNase P1, and 7.3 pmol/ml [3H]RNA for 2 h at
37 °C. 15.4 µl of 1 M Tris buffer, pH 8.0, and 5.4 units of bacterial alkaline phosphatase were added, the volume was
adjusted to 275 µl, and the incubation continued for an additional 2 h at 37 °C. The reaction was stopped by the addition of
acetic acid to a concentration of 27 mM. m2G
was added as an internal standard, and the mixture was analyzed by HPLC
(11).
Protection Studies--
[3H]Methyl-labeled 16 S
RNA (0.36 pmol) was hybridized with a 50-fold excess of
deoxyoligonucleotide in a 50-µl reaction mixture, which also
contained 40 mM Mes, pH 6.4, 400 mM NaCl, 9 mM EDTA, and freshly deionized 80% (v/v) formamide. The
samples were heat-denatured at 90 °C for 10 min and then placed at
room temperature for 15 min. They were then diluted with 9 volumes of
ice-cold RNase Buffer (10 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 5 mM EDTA), and RNase T1 (0.2-1.8
Sankyo units) was added. The samples were digested for 30 min at
30 °C. Digestion was stopped by the addition of 9 volumes of cold
10% trichloroacetic acid. After 10 min on ice, the samples were
filtered on BA85 cellulose nitrate filters, the filters were dissolved,
and the samples were counted.
Other Methods--
N-terminal sequencing was performed on
samples of the purified enzyme, which had been electrophoresed on SDS
gels. The protein was electroblotted onto a polyvinylidene difluoride
membrane following standard procedures (17), and N-terminal sequencing
was performed as described previously (16). Protein content was assayed
by a modified Bradford procedure (Bio-Rad protein assay, catalog number
500-006) using bovine serum albumin as standard. SDS gels were 12%
polyacrylamide and contained 0.38 M Tris-HCl, pH 8.8, and
0.1% SDS. The 5% stacking gel contained 0.13 M Tris-HCl,
pH 6.8, and 0.1% SDS. Samples were heated at 95 °C for 5 min in
Buffer LB and then quenched on ice for 5 min before loading. Gels were stained with Coomassie Blue.
Purification of the Methyltransferase and Identification of the
Gene--
Previous work from this laboratory had identified two
E. coli 16 S RNA methyltransferases, one specific for
m2G966 and another specific for m5C967 (11, 13,
14). The m2G966 enzyme reacted with unmodified 30 S
subunits (11, 13) or unmodified 16 S RNA complexed with 30 S proteins
S7 and S19 (14) but not with free 16 S RNA, whereas the
m5C967 enzyme had the reciprocal specificity. The purpose
of the present work was to identify and clone the gene for the
m2G methyltransferase. Following a purification scheme
similar, but not identical, to that previously used, a preparation that reacted with 30 S but not 16 S RNA was obtained. Gel electrophoresis showed a single strong band at ~37 kDa. N-terminal sequencing of this
band yielded the following sequence: SAFTPASEVLLRHSDDFEQ. A search of
the GenBank data base showed an exact match to ORF yjjT
(SWISS-PROT accession number P39406). The calculated molecular mass was
37.6 kDa, in good agreement with the experimental value. The presence
of a strong 7-nucleotide Shine-Dalgarno sequence 6 nucleotides upstream
from the initiating ATG was observed.
Cloning of the Gene, Overexpression, and Affinity
Purification--
The putative gene was cloned into pET-15b by
standard methods. Induced cells containing clones with this insert
produced large amounts of protein ~37 kDa in size, whereas no such
band was visible in the pET control (data not shown). The overexpressed
protein was distributed between the S15 supernatant fraction and S15
pellet. Distribution between these two fractions varied somewhat from preparation to preparation, and renaturation of the enzyme in the
pellet produced variable results. Therefore, all experiments were
conducted using the soluble supernatant fraction of the enzyme. This
fraction was purified by affinity chromatography on a
Ni2+-containing resin, because the overexpressed protein
contained an N-terminal His tag. The eluted protein product gave a
single band on gel electrophoresis at the expected size (data not
shown). The finding of methyltransferase activity in the
affinity-purified protein (see below) is definitive proof that the gene
cloned is a methyltransferase gene.
Substrate and Product Specificity of the m2G
Methyltransferase--
The overexpressed enzyme possessed the same
preference for 30 S particles over free RNA as described previously
(14) for the partially purified m2G966 methyltransferase
(Fig. 1). The m2G enzyme was
able to incorporate ~0.5 pmol of methyl groups/pmol of particles in
20 min, whereas 0.02 pmol of methyl groups was incorporated/pmol of
free 16 S RNA in the same time interval. The nature of the methylated
nucleoside was examined by HPLC. [3H]methyl 30 S
ribosomes were prepared using the recombinant enzyme, and the 16 S RNA
was isolated. After digestion to nucleosides with RNase P1 and alkaline
phosphatase, the mixture was analyzed by HPLC (Fig.
2). A single peak containing 90% of the
radioactivity loaded onto the column was eluted at the m2G
position, as confirmed by its co-elution with the added internal marker
of m2G (Fig. 2, arrow). These results confirm
that the recombinant enzyme does catalyze formation of m2G
in 16 S RNA.
Localization of the Site of Methylation--
E. coli16 S RNA
contains three m2G residues, at positions 966, 1207, and
1516 (Ref. 18 and Fig. 3). To determine
which G was methylated by the recombinant enzyme,
hybridization-protection studies were conducted using deoxyoligomers
that were complementary to the RNA sequence spanning each of the
m2G sites (13). Oligomer 958 spanned the region from
residue 958 to residue 977, which included m2G966; oligomer
1197 spanned the region from residue 1197 to residue 1216, including
m2G1207; and oligomer 1506 spanned the region from residue
1506 to residue 1525, which included m2G1516 (Fig. 3). The
m2G methyltransferase previously characterized was specific
for G966 (13). Because the purification scheme for the native enzyme used in this work was similar, and the recombinant enzyme had the same
substrate specificity, we supposed that the same methyltransferase had
been cloned. Unexpectedly, the hybridization-protection experiment showed clearly that this was not the case (Fig.
4). Oligomer 958 was as ineffective in
protecting the label in the RNA from RNase digestion as was oligomer
1506 or no oligomer. On the other hand, oligomer 1197 efficiently
protected the RNA from degradation. To control for artifacts, the
deoxyoligonucleotides used in this experiment were the identical
preparations used previously (13). In that work, oligomer 1197 did not
protect either the m2G- or
m62A-containing RNAs. Therefore, its protection
here cannot be attributable to some generalized inhibitory effect on
RNase T1. In that work, oligomer 958 did protect both the
m5C and m2G labels; thus its failure to protect
here cannot be attributable to some failure of hybridization. Likewise,
because oligomer 1506 did protect the
m26A-containing RNA, its inactivity here cannot be
attributable to a hybridization failure. We conclude from these results
that the recombinant m2G methyltransferase described here
specifically modifies G1207 rather than G966.
Mg2+ Dependence of the m2G
Methyltransferase--
Because of the fact that this methyltransferase
recognized 30 S ribosomes but not free RNA, we expected that
Mg2+ would be required for this reaction, even though a
similar methyltransferase specific for m5C967 and for 16 S
RNA functioned equally well in 1 mM EDTA as in 10 mM Mg2+.2 Moreover, because the
m2G966 methyltransferase studied previously was inhibited
in its methylation of 30 S subunits by increasing concentrations of
Mg2+ (14), we tested two concentrations, 0.9 mM, which should cause partial unfolding of the particle,
and 6 mM, which is known to be sufficient to stabilize
natural 30 S particles in this buffer. Fig.
5 shows that at the lower
Mg2+ concentration, methylation approaches unit
stoichiometry (0.93 mol/mol), whereas at 6 mM
Mg2+, the reaction levels off at ~0.3 mol of methyl/mol
of ribosomes. Some Mg2+ is required, however, because in
other experiments, 2 mM EDTA decreased the plateau level
from 0.74 mol of methyl incorporated/mol of 30 S in 0.9 mM
Mg2+ to 0.14 mol/mol in EDTA. In these experiments, the
substrate was not preincubated under the specified Mg2+
conditions before the methylation reaction was initiated. In a single
preincubation experiment in which the ribosomes were incubated in
reaction buffer without SAM for 10 min at 37 °C before initiation of
the reaction with SAM, the presence of 2 or 10 mM EDTA
reduced incorporation levels further to 0.1 or <0.1 mol of methyl/mol
of ribosomes, respectively, whereas the control in 0.9 mM
Mg2+ still yielded 0.8 mol/mol.
Substrate Specificity and Mg2+ Requirements of the
Enzyme--
Because the m2G1207 methyltransferase reacts
with 30 S particles but barely at all with 16 S RNA, it seems likely
that methylation of the G residue occurs after the 16 S RNA has
associated with some ribosomal proteins. The m2G966
methyltransferase, which has similar specificity, has been shown to
only require the presence of proteins S7 and S19 with the 16 S RNA to
be recognized by the enzyme (14). Whether the m2G1207
methyltransferase can also efficiently recognize 16 S RNA complexed
with only a few ribosomal proteins, and what these proteins are, must
await further study.
The Mg2+ requirements for methylation are also consistent
with the hypothesis that G1207 is methylated after the association of
an unspecified number of ribosomal proteins but before assembly of the
30 S subunit is complete. At 0.9 mM Mg2+,
virtually all of the G1207 residues in the 30 S subunit were available
for reaction, whereas when the Mg2+ concentration was
raised to 6 mM, only
The substrate preference for 30 S ribosomes versus free RNA
shown here for the m2G1207 enzyme and previously for the
m2G966 methyltransferase (13, 14) is shared by the KsgA
enzyme, which also requires a 30 S subunit for reaction (20). Perhaps the ability of the m5C967 methyltransferase to react with
free RNA, but not with 30 S subunits, is the exception to the rule, and
most or all of the other 16 S RNA-modifying enzymes require either a
ribonucleoprotein or a complete 30 S subunit. RsuA, which makes the
single pseudouridine in 16 S RNA, also has a specific requirement for a
particular RNP particle (15).
How did two similar purification procedures yield an m2G966
enzyme previously and an m2G1207 enzyme in the current
work? We suspect this occurred during the DEAE column purification
step. Previously the eluate was only assayed with free RNA in the
belief that the m2G activity, measured using 30 S subunits,
co-purified at that step with the m5C activity assayed with
free RNA. However, in the present work the DEAE eluate was assayed
directly with 30 S. It is possible that the m2G966 enzyme
was overlooked in the latter case, and in the former that the
m2G1207 enzyme did not exactly co-purify with the
m5C enzyme. Whatever the explanation, the sequenced protein
identified the only ORF in E. coli with that N-terminal
sequence, and this proved to be the m2G1207 methyltransferase.
Gene and Protein Sequence--
The gene coding for the
m2G1207 methyltransferase is yjjT. This ORF
codes for a 343-amino acid protein with a calculated molecular mass,
37.6 kDa, that agrees well with the value of 37 kDa for the native
enzyme. We propose that the ORF be renamed rsmC for (r)ibosomal (s)mall subunit (m)ethyltransferase, because the function of the gene product has been identified as the enzyme for formation of
m2G1207. C denotes that this is the third gene sequence for
an rRNA methyltransferase to be described, the first one being
ksgA, the gene for the m26A
methyltransferase (8), and the second one being rsmB, the gene for the m5C967 methyltransferase.2 In
keeping with this nomenclature, we suggest that rsmA would be a suitable alternate name for ksgA.
The RsmC sequence contains the SAM-binding motif
DXGXGXGXL (21) at residues
201-209 as well as the somewhat longer motif in which this is
embedded, which has been described by Koonin et al. (22) at
residues 199-215. A search of the GenBank data base using BLAST 2.0.4 (23) identified proteins with highly significant similarity to the
m2G1207 methyltransferase in seven organisms. These
included the prokaryotes E. coli, Hemophilus
influenzae, Bacillus subtilis, Staphyloccus
aureus, and even the extremely divergent Thermotoga neapolitana as well as the archaebacteria Pyrococcus
horikoshii and Methanococcus jannaschii. Somewhat less
related were proteins from Streptomyces anulatus, Streptomyces
pristinaespiralis, and Chlamydia trachomatis. No strong
similarities were found with any higher eukaryotic ORFs.
The protein most similar in sequence to RsmC according to this analysis
is from H. influenzae (P44453, HI0012). This protein is 330 amino acids long compared with 342 for RsmC. They are 46% identical
and 65% similar along 328 residues. Given the high level of similarity
between the two sequences, it is reasonable to suppose that this gene
codes for an equivalent methyltransferase in H. influenzae
and, therefore, that this organism also has an equivalently located
m2G in its 16 S RNA. No information is available as to the
presence of such an m2G in H. influenzae or for
that matter in any of the other prokaryotes or archaebacteria. The next
most similar amino acid sequence to RsmC is the hypothetical protein
product of the E. coli ygjO gene. This 43.4-kDa protein is
33% identical and 50% similar over 173 residues to RsmC. We suspect
that this protein is the m2G966 methyltransferase
that we described previously (13, 14).
Role of Methylation of G1207 in the
Ribosome--
m2G1207 is located in the helix 34 stem (see
Fig. 3) in a region believed to be involved in recognition of peptide
chain termination codons (24, 25). This region has been directly linked
to the decoding site on the ribosome by cross-links from U1052, which is adjacent to m2G1207 on the opposite strand to the A site
codon of mRNA (26) and from A1196, 11 nucleotides away on the same
strand, to the next downstream mRNA codon (10). However, no
specific role for m2G1207 is known in chain termination or
codon recognition or, for that matter, in any other function of the
ribosome. Moreover, it cannot be essential for ribosome function
because, as noted in the introduction, functional 30 S ribosomes have
been prepared lacking all modified nucleosides. However, as also noted
there, the assembly ability as well as function relative to modified controls was reduced.
To study the reasons for this reduction in assembly and function, we
have embarked on a program to identify all of the genes responsible for
the modifying enzymes that act on E. coli 16 S RNA. So far,
we have identified three such genes, rsuA (15), rsmB,2 and in this work, rsmC. rsmA
(ksgA) was already known (8). There should be at most seven
more genes to identify. Two putative genes have already been identified
by their strong sequence similarity to RsmB and RsmC, respectively.
Gene inactivation, one or more at a time, to block selected
modifications should then help unravel the mystery of what modified
nucleosides do in the ribosome.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
20 °C.
13 to
+14, where A of the initiating ATG is +1, with changes at
1,
2, and
3 to create an NdeI site adjacent to the initiating ATG.
The C-terminal primer, in the reverse orientation, extended from +1061
to +1087, where the last sense nucleotide is 1029, and contained
mismatches at 1073 and 1074 to create a BamHI site. After
polymerase chain reaction, the amplified product was isolated by
agarose gel electrophoresis, extracted using the Wizard DNA
purification kit, concentrated by membrane filtration (Amicon Microcon
100), and digested with NdeI and BamHI. The
pET-15b vector, also digested with NdeI and BamHI, and the gene insert were ligated by standard methods
using T4 DNA ligase. Transformation of XL1-Blue Epicurian cells was performed by standard methods. Plasmids from three clones containing the insert were transferred into BL21/DE3 cells.
-D-thiogalactopyranoside at 1 mM
was added to the culture, and incubation continued at 37 °C to an
A600 of 1.4-1.8. Cells were recovered by centrifugation and frozen at
70 °C. For analysis of the whole cell contents, cells from 1 ml of culture were suspended in 0.03 ml of 10 mM Tris, pH 8, plus an equal volume of 2 × Buffer LB,
heated to 100 °C for 5 min, and then chilled. For preparation of
recombinant protein, cells from 100 ml of culture were washed in 10 mM Hepes, pH 7.5, and 0.17 M NaCl and sonicated
in 6 ml of either 20 mM Tris, pH 8, 20 mM
NH4Cl, 5 mM MgCl2, 0.1 mM EDTA, 5 mM BME, and 1 mM
phenylmethanesulfonyl fluoride or Binding Buffer plus 1 mM phenylmethanesulfonyl fluoride. Both buffers gave equivalent results. The sonicated mixture was centrifuged at 15,000 × g to
obtain the S15 supernatant and pellet fractions. The S15 pellet was
dissolved in 6 ml of Buffer D or in Binding Buffer containing 6 M urea.
20 °C.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Substrate Specificity of the recombinant
m2G methyltransferase. In vitro transcripts of
16 S RNA ( ) and 30 S ribosomes (
) reconstituted from this
transcript and a complete set of 30 S ribosomal proteins were prepared,
and methylation was measured as incorporation of 3H into a
trichloroacetic acid precipitate. The concentrations of 16 S RNA and 30 S particles were 100 and 80 nM, respectively. The
Mg2+ concentrations were 4 mM (
) and 2 mM (
). The purified enzyme protein concentration was
0.41 µg/ml.
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Fig. 2.
Characterization of the methylation product
of recombinant m2G methyltransferase.
[3H]methyl-reconstituted 30 S ribosomes were prepared
using [3H]methyl-labeled SAM and recombinant
m2G methyltransferase. 1.05 pmol of CH3/pmol of
30 S were obtained. The RNA was extracted with phenol, isolated,
digested with RNase P1 and alkaline phosphatase, and analyzed by HPLC.
The position of the m2G added as an internal standard was
determined by its UV absorption and is marked by the
arrow.
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Fig. 3.
Location of the m5C,
m2G, and m26A residues and the sites of
complementary deoxyoligonucleotides used in the
hybridization-protection experiments. The positions of
m5C, m2G, and m26A
(arrows) are shown superimposed on the secondary structure
of E. coli 16 S RNA (19). The segments of RNA complementary
to the deoxyoligomers used in the hybridization-protection experiments
are boxed.
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Fig. 4.
Localization of the site of m2G
formation by the recombinant methyltransferase.
[3H]Methyl 16 S RNA (0.36 pmol) extracted from 30 S
ribosomes that had been labeled (1.05 pmol of CH3/pmol of
30 S) using the recombinant m2G methyltransferase was
hybridized with the indicated oligonucleotide and then digested with
RNase T1 (0.2-1.8 Sankyo units) as described under "Experimental
Procedures." , oligomer 1197-1216;
,
oligomer 958-977A;
, oligomer 958-977B;
,
oligomer 1506-1525;
, no oligomer. Oligomer 958-977A
was a fresh dilution from the original stock solution (11), and
oligomer 958-977B was the same dilution used previously (11,
13).
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Fig. 5.
Mg2+ dependence of methylation by
the recombinant m2G methyltransferase. Methylation of
reconstituted 30 S ribosomes was conducted as described under
"Experimental Procedures," except that the incubation contained 0.9 ( ) or 6 (
) mM Mg2+. The 30 S ribosome
concentration was 40 nM and the purified enzyme
concentration was 0.45 µg/ml.
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
were still available. On
the other hand, in EDTA, only
were reactive, and
preincubation reduced that amount to less than
. The
structure of the 30 S particle at 0.9 mM Mg2+
is probably partially disordered and thus may mimic the structure of
the ribonucleoprotein intermediate, which may be the true substrate. In
EDTA, this structure may be lost, and at 6 mM
Mg2+, the 30 S particle may be too compact for reactivity.
The observed partial reaction at 6 mM Mg2+ may
be attributable to the fact that 30 S ribosomes assembled from an RNA
transcript are less stable than ones reconstituted from natural RNA (4,
16). We emphasize a role for Mg2+ in maintaining the proper
substrate structure rather than a role at the catalytic center of the
enzyme solely by analogy with the m5C967 methyltransferase,
which had no Mg2+ requirement.2 Clearly, the
current experiments do not allow us to distinguish between the two possibilities.
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FOOTNOTES |
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* This work was supported by funds from Hoffmann-La Roche, Inc., and a Markey Foundation grant to the Department of Biochemistry and Molecular Biology, University of Miami School of Medicine. Experimental work was performed at the now defunct Roche Institute of Molecular Biology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Science and Technology, Bergen Community
College, Paramus, NJ 07652.
§ Present address: RNA Research Group, Dept. of Molecular Recognition, Smithkline Beecham Pharmaceuticals, 709 Swedeland Rd., King of Prussia, PA 19406.
¶ Present address: Tarrant County College, Arlington, TX 76018.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, University of Miami School of
Medicine, Miami, FL 33101. Tel.: 305-243-3677; Fax: 305-243-3955.
The abbreviations used are: SAM, S-adenosyl-methionine; HPLC, high performance liquid chromatography; ORF, open reading frame; Mes, 4-morpholineethanesulfonic acid.
1 L. Niu and J. Ofengand, unpublished results.
2 J. S. Tscherne, K. Nurse, P. Popienick, H. Michel, M. Sochacki, and J. Ofengand, submitted for publication.
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