From the Laboratoire d'Enzymologie et Biochimie
Structurales, CNRS, Gif-sur-Yvette, France and the
§ Universität Heidelberg, Institut für Biochemie
I, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The enzymatic activity of yeast gene product Deg1
was identified using both disrupted yeast strain and cloned recombinant protein expressed in yeast and in Escherichia coli. The
results show that the DEG1-disrupted yeast strain lacks
synthase activity for the formation of pseudouridines 38
and
39 in tRNA whereas the other activities, specific
for
formation at positions 13, 27, 28, 32, 34, 35, 36, and 55 in
tRNA, remain unaffected. Also, the His6-tagged recombinant
yeast Deg1p expressed in E. coli as well as a protein
fusion with protein A in yeast display the enzymatic activity only
toward
38 and
39 formation in different
tRNA substrates. Therefore, Deg1p is the third tRNA:pseudouridine
synthase (Pus3p) characterized so far in yeast. Disruption of the
DEG1 gene is not lethal but reduces considerably the yeast
growth rate, especially at an elevated temperature (37 °C). Deg1p
localizes both in the nucleus and in the cytoplasm, as shown by
immunofluorescence microscopy. Identification of the pseudouridine
residues present (or absent) in selected naturally occurring
cytoplasmic and mitochondrial tRNAs from DEG1-disrupted
strain points out a common origin of
38- and
39-synthesizing activity in both of these two cellular compartments. The sensitivity of Pus3p (Deg1p) activity to overall three-dimensional tRNA architecture and to a few individual mutations in tRNA was also studied. The results indicate the existence of subtle
differences in the tRNA recognition by yeast Pus3p and by its
homologous tRNA:pseudouridine synthase truA from E. coli (initially called hisT or PSU-I gene
product).
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The modified nucleoside pseudouridine
(5-(-D-ribofuranosyl)uracil, abbreviated as
),1 is found very
frequently in all kinds of RNA from eubacteria, archaea, and eukaryotes
(1). It is present in all transfer RNAs (2), large and small subunits
of ribosomal RNA (3, 4), most small nuclear RNAs (5, 6), and selected
small nucleolar RNAs (7).
The numerous residues in RNA are produced by a family of enzymes
(pseudouridine synthases, RNA:
synthases). These enzymes act on
specific uridine residues of the RNA molecules, but still very little
is known about the number of these enzymes in a given cell as well as
their mechanism and their RNA recognition mode.
In yeast tRNA, formation of pseudouridines at positions 13, 32, and 55 is catalyzed by three distinct enzymes (8), whereas in the same yeast
cell one single enzyme (Pus1p) is responsible for formation at
positions 27, 34, and 36 (9). Several distinct pseudouridine synthase
activities acting on eukaryotic small nuclear RNAs were also detected
in crude cell extracts (5); however, none of these enzymes has been
identified so far. From recent works on rRNA maturation, it appears
that most (if not all) of the
in eukaryotic rRNA is probably
synthesized by a single (or at least very few) rRNA:
synthase(s); in
this latter case the enzyme(s) is(are) guided to the different target
uridines within the rRNA by a huge family of diverse small nucleolar
ribonucleoproteins present in the nucleolus (10, 11; for review, see
Ref. 12).
The first RNA:pseudouridine synthase that was purified and fully
characterized came from Salmonella typhimurium (13) and later from Escherichia coli (14). It corresponds to a
tRNA: synthase (PSU-I, previously called hisT gene
product, recently renamed to truA). It catalyzes the
formation of
38,
39, and/or
40 in several cellular tRNAs. Only the gene for the
E. coli enzyme (PSU-I) was cloned and sequenced (15). The
E. coli PSU-Ip is a monomer with a molecular mass of 31 kDa
(270 amino acids), whereas PSU-Ip from S. typhimurium has a
subunit size of about 50 kDa and dimerizes in the presence of tRNA
substrate (13). A higher eukaryotic homolog of PSU-I from calf thymus
was highly enriched after five chromatographic steps (16); however, a
homogeneous preparation was not obtained. All of these enzymes have
similar properties: they do not require any cofactor, they catalyze the
formation at contiguous sites (region specificity), and they demonstrate similar kinetic properties (13, 14, 16).
Recently three other E. coli RNA: synthases were
identified and their corresponding genes cloned: truB, which
is site-specific for
at position 55 in tRNA (17); rluA,
specific for
at position 32 in tRNA and at position 746 in 23 S
rRNA (dual specificity; Ref. 18); and rsuA, specific for U
at position 516 in 16 S rRNA associated with ribosomal proteins within
a ribosomal ribonucleoprotein complex (19).
The genes for two other tRNA:pseudouridine synthases (Pus1p and Pus2p)
from yeast were also identified, one of them (PUS1) was
cloned and overexpressed in E. coli (9). The recombinant yeast Pus1p was shown to be specific for at position 27 in several yeast tRNAs and for
at positions 34 and 36 in the intron-containing pre-tRNAIle (9; for review, see Ref. 20). This enzyme has a
molecular mass of 62 kDa and was shown to be located essentially in the
yeast nucleus (9). The target RNA and position(s) of uridine to be modified by Pus2p (a 42-kDa protein) have not been determined yet.
Another yeast protein has been reported to be homologous to E. coli and S. typhimurium pseudouridine synthases PSU-I
(hisT gene product, truAp) (21). This protein (called Deg1
because disruption of the gene causes depressed growth; SacchDB
accession number YFL001W, SwissProt P31115) also displays a significant homology to the yeast Pus1p (9). Based on this sequence homology with
PSU-I, it was suggested (21) that Deg1 protein may possess the
corresponding activity of hisT gene product, but this
plausible hypothesis was never tested experimentally. Here we present
experimental evidence that yeast protein Deg1 is indeed a new distinct
yeast tRNA: synthase (Pus3p) with a slightly different specificity toward tRNA compared with E. coli enzyme truA since it
catalyzes the formation of only
38 and
39
(not
40). The structural substrate requirements and the
intracellular localization of Pus3p are also analyzed.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chemicals, Enzymes, and
Materials--
-32P-Radiolabeled nucleotide
triphosphates (400 Ci/mmol) were from Amersham (U. K.). Tris,
dithioerythritol, dithiothreitol, nucleoside triphosphates,
Penicillium citricum nuclease P1, Aspergillus oryzae RNase T2, and phenylmethylsulfonyl fluoride were from
Sigma. Diisopropyl fluorophosphate and CMCT were from Aldrich.
Bacteriophage T7 RNA polymerase, restriction enzymes, and
isopropyl-1-thio-
-D-galactopyranoside were from from MBI
Fermentas (Vilnius, Lithuania). RNasin and avian myeloblastosis virus
reverse transcriptase were from Promega. Synthetic
oligodeoxynucleotides used as primers for reverse transcription were purchased from MWG-Biotech (Germany) and used without further purification. Thin layer cellulose plates were from Schleicher & Schuell (Germany), and all other chemicals were from Merck Biochemicals (Germany).
Plasmids and Transcription of tRNA Genes-- The plasmids carrying the synthetic genes of tRNAs used in this work were described previously tRNAAsp (GUC) (22), pre-tRNAIle (UAU) and its mutant (anticodon UUA) (23), pre-tRNATyr (mutant UUA) (24), and E. coli tRNASer (GGA) (25). Plasmids with cloned genes of yeast tRNAPhe (GAA) and its mutants PheY54 (p67YF1, mutation C56G) and PheY55 (p67YF2, mutations G19C and C56G) have been described (26). Plasmids carrying yeast tRNAHis (GUG), yeast tRNASer (IGA), and mutant yeast tRNAVal (harboring anticodon CAU instead of UAC) were gifts, respectively, of Dr. J. Rudinger (IBMC, Strasbourg), Dr. H. Himeno (Tokyo University, Japan), and Dr. F. Fasiolo (IBMC). The synthetic gene for yeast tRNAAla (anticodon IGC) was constructed by ligation of sets of complementary oligonucleotides as described (22).
The DNA template for T7 transcription of E. coli tRNALeu (CAG) and yeast intronless tRNATrp (bearing a mutated anticodon CUA) were prepared by PCR amplification of the corresponding sequence present in E. coli genomic DNA and in plasmid (pT7T3am2Yeast Strains, Media, and Microbiological Techniques--
The
wild type yeast strain used in this study was RS453 (/a,
ade2/ade2, leu2/leu2, ura3/ura3, his3/his3, trp1/trp1), and all
mutant strains containing disrupted genes were derived from this one.
Yeast cells were grown on minimal SDC and rich YPD medium (37), and
sporulation of diploid cells on YPA plates and tetrad analysis were
performed according to Ref. 29. Minimal SDC medium/plates were
supplemented by all amino acids and nutrients except the ones used for
the selection or, if indicated, contained 5-fluoroorotic acid (CSM
medium, BIO 101, La Jolla). Genetic manipulations were performed as
described (30).
Cloning and Expression of Yeast Deg1p in E. coli--
Preparation of the construct for expression of Deg1 in yeast
was done as follows. The DEG1 gene was amplified by PCR from total yeast genomic DNA using two primers that created an
XbaI restriction site in the 5-untranslated region of the
gene 151 nucleotides upstream of the start codon
(ttttttctagAATCAATGGGCTCAGCTC, complementary sequence in capital
letters) and an SalI restriction site
(tttttgtcgacAAGAAATATAGTCTTCAAGG) in the 3
-untranslated region of the
gene 50 nucleotides downstream of the stop codon. This allowed cloning
of the gene into a pRS315 vector previously cut with
XbaI/SalI. This construct could complement the
slow growing phenotype when transformed in the
deg1
haploid cells.
Gene Disruption of DEG1-- Disruption of the DEG1 gene was done by the one-step gene replacement method (31). In this study, the DEG1 gene was disrupted by inserting a BamHI fragment 0.9 kilobases long and containing the HIS3 gene into the BamHI site of the DEG1-ORF cloned in the pET8c vector. The disrupted gene was excised and the linear fragments used to transform the diploid strain RS453. HIS+ transformants with the correct integration of the interrupted gene at the DEG1 locus were verified by PCR analysis (data not shown). Correct integrants were sporulated, and tetrads were dissected. A 4:0 segregation for viability and a 2:2 segregation for the HIS marker were found for the DEG1 gene disruption showing that this gene is not essential for cell growth. However, all spores carrying a disrupted DEG1 gene grew slower giving rise to small colonies as reported previously (21).
Construction of Doubly Disrupted Mutants--
To construct a
haploid yeast strain in which the disrupted PUS1 and
DEG1 genes are combined, a PUS1
mutant harboring pURA3-PUS1 was mated to the mutant
DEG1
. The resulting heterozygous diploids were
sporulated, and tetrad analysis was performed. For complete tetrads in
which the HIS+/his
genotype segregates 2:2, one can predict that the two HIS+
progeny are
deg1::HIS3/pus1::HIS3.
A complete tetrad showing this segregation pattern was analyzed in
greater detail for the segregation of the HIS3 and
URA3 markers by plating cells on SDC-his and SDC-ura plates,
respectively. The HIS+ progeny
deg1::HIS3/pus1:HIS3
also contained the plasmid pURA3-PUS1; we could shuffle out this
plasmid and test whether the double mutant
pus1
/deg1
gives
synthetic lethality by plating this strain on 5-fluoroorotic acid
containing plates at 30 °C. The
los1
/deg1
strain was constructed
in a similar way.
Construction of the Deg1p Fusion Protein Carrying Protein A or Green Fluorescent Protein (GFP) as a Tag-- Epitope tagging of Deg1p was done by fusing two IgG binding units from Staphyloccus aureus protein A to the N-terminal end of Deg1p. For this gene fusion, a new PstI restriction site was generated at the ATG codon of DEG1 by PCR-mediated mutagenesis, and the ORF was subcloned into the plasmid pRS315 in-frame with the two IgG binding units under the control of the NOP1 promoter (PNop1-ProtA cassette; see Ref. 32), creating the plasmid pNOP-ProtA-Deg1. Affinity purification of the ProtA fusion protein was done as described previously (32). Tagging with GFP was done in a similar way, but the IgG binding units were replaced by the ORF for GFP (PNop1-GFP cassette),2 creating the plasmid pNOP-GFP-Deg1. The GFP used is a S65T/V163A variant exhibiting enhanced fluorescence properties (33, 34).
Cellular Localization of DEG1 Gene Product-- Intracellular localization of the ProtA-Deg1p fusion protein was performed by indirect immunofluorescent microscopy as described in Ref. 9 using as first antibody rabbit anti-protein A (Sigma) and as second antibody CyTM3-conjugated AffiniPure donkey anti-rabbit IgG (Dianova). GFP-Deg1p was observed in living cells by direct fluorescent microscopy.
Pseudouridine Formation Assay in Vitro--
Preparation of S100
extract from yeast as well as from E. coli was made as
described elsewhere (25, 28). The activity of yeast extracts and
purified yeast enzyme fractions was tested at 30 °C; 37 °C was
used for testing E. coli extracts. The incubation mixture
contained 100 mM Tris-HCl, pH 8.0, 100 mM
ammonium acetate, 5 mM MgCl2, 2 mM
dithiothreitol, 0.1 mM EDTA, and 1-2 fmol of 32P-radiolabeled T7 runoff transcripts as substrate. After
incubation, the pseudouridine content in the radiolabeled transcripts
was analyzed as described previously (27, 28). In brief, the RNA was
first extracted with phenol-chloroform, precipitated in ethanol, and
then hydrolyzed completely to 3-nucleotide monophosphates by RNase T2.
Each hydrolysate was chromatographed on two-dimensional thin layer
chromatography plates, and the radioactivity in the
MP and UMP spots
was evaluated after exposing the thin layer chromatography plates with
a PhosphorImager screen. Taking into account the relative number of
MP and UMP in the tRNA molecule, the relative amount of
over
tRNA molecule (expressed in mol/mol of tRNA) can be evaluated. The
accuracy of this method was found to be about ± 0.05 mol of
/mol of tRNA.
Identification and Localization of Naturally Occurring Residues in tRNAs--
Localization of pseudouridine residues in tRNA
was performed as described (35, 36) with the following modifications.
10 µg of total tRNA extracted from wild type or mutant yeast strain was treated by 0.17 M CMCT in Bicine-urea buffer, pH 7.5, for 15 min at 42 °C. Reverse transcription was done at 42 °C for
45 min using about 1 µg of CMCT-modified cytoplasmic tRNA or 3 µg of CMCT-modified mitochondrial tRNA and 1-2 pmol of
5
-32P-labeled synthetic oligodeoxynucleotide primer.
Reverse transcription products were separated on 15% denaturing
polyacrylamide gel. The oligodeoxynucleotide primers were chosen to be
complementary to the 18 nucleotides at the 3
-end of cytoplasmic
tRNAGly and mitochondrial tRNAArg,
respectively, because both of these tRNAs have a low content of
modified nucleotides downstream from the pseudouridylation sites.
Miscellaneous-- Isolation of total yeast DNA was done essentially as described in Ref. 37. DNA and plasmid manipulations (restriction analysis, end filling reactions, ligations, PCR amplifications, DNA fragment recovery, and small scale and large scale plasmid preparations) were done essentially according to standard procedures (38). The nucleotide sequence of tRNA inserts in the various plasmids used was verified systematically by the dideoxy sequencing technique. Evaluation of the protein concentration was done according to Bradford (39), and Western blotting was performed as described in Ref. 9.
Screening of the nonredundant GenBank data base was performed using BLAST algorithm, version 1.4.9; amino acid substitution matrix BLOSUM62 was used in all data base searches. Multiple sequence alignment was constructed using Macintosh version of MACAW software, version 2.0.5. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Yeast Protein Deg1 Displays Significant Sequence Homology with E. coli truA and Yeast Pus1--
The accessibility of whole genome
sequencing data for several prokaryotic and eukaryotic organisms allows
the systematic screening for the corresponding enzymes based on
sequence homology. BLAST search, using the
hisT(truA) gene of E. coli (coding for
tRNA:38-40 synthase; see Ref. 15) as a query sequence,
allows detection of several homologous proteins in the GenBank. Partial
sequence alignment of hisT(truA)-like proteins of
different origin is presented on Fig. 1.
These are mostly putative pseudouridine synthases I (PSU-I) from
several bacteria and lower eukaryotes. Higher eukaryotes are presented
by the partial nucleotide sequence of human (zn81c07.s1) Deg1-like
protein. All of these putative proteins display significant sequence
homology with E. coli truA (BLAST p value is less
than 0.001). All proteins share common signature blocks of amino acid, which suggests the same or similar function in the cellular metabolism. Here it is noteworthy to remember that the yeast PUS1 gene
also shares high homology with the truA(hisT)
gene, but the corresponding RNA:
synthase has a different
specificity and properties (9). Also, as already noted by Koonin (40),
this group of hisT(truA)-like enzymes is rather
distant from the pseudouridine synthases identified so far, specific,
respectively, for
55 (E. coli truB-like), for
32 in tRNA and
746 in 23 S rRNA
(E. coli rluA-like), and for
516 in E. coli 18 S rRNA. From the above sequence homology one can expect
that protein Deg1 has enzymatic activity similar to that of E. coli truA.
|
Disruption of Deg1 Gene Results in the Disappearance of
tRNA:38/39 Enzymatic Activity in the Yeast Cell
Extract--
To test the function of yeast Deg1 protein, we performed
one-step gene disruption of the corresponding gene in yeast. The resulting yeast strain remains viable, but the deg1
mutant grows slower compared with parent wild type strain cells as was
reported previously (21). Furthermore, we observed that the slow growth
phenotype is particularly strong at 37 °C (Fig. 2). This thermosensitive phenotype and
growth defect of the deg1
strain could be fully
complemented by expressing the cloned DEG1 gene or the
fusion proteins ProtA-Deg1p and GFP-Deg1 (Fig. 2 and below).
|
|
|
38/39 Synthase Activity Resides in the Deg1
Protein--
To show that Deg1p is solely responsible for the
modifications missing in the DEG1-disrupted strain, the
recombinant protein was expressed in E. coli and its
activity tested in vitro. The results show that the activity
of tRNA:
38/39 synthase is readily detected in the
extract of E. coli expressing His6-Deg1 and also upon the fractionation of the induced extract on
Ni2+-nitrilotriacetic acid-agarose. Activity of the
expressed yeast pseudouridine synthase was retained considerably by
metal affinity column, and activity toward
38 in yeast
tRNAAla was detected only in the case of induced E. coli extract. Similar results were obtained using purified by
S-Sepharose FF, hydroxyapatite, and Ni2+-nitrilotriacetic
acid-agarose column recombinant Deg1p (more than 95% purity), which
efficiently catalyzes the pseudouridine formation in the transcripts of
yeast tRNAPhe (
39) and tRNAAla
(
38) (data not shown).
|
Specificity of Yeast and E. coli tRNA:38-40
Synthases Is Different--
We compared the specificity of the yeast
and E. coli enzymes using the transcripts of various yeast
and E. coli tRNAs. Fig. 5
shows the anticodon stem-loop regions of the naturally occurring tRNAs,
the transcripts of which were tested for the formation of
38,
39, and
40. As shown
in Fig. 6A (see also Table
II), the transcript of yeast
tRNAHis (anticodon GUG) is modified efficiently to
pseudouridine at position 39 upon the incubation in yeast or E. coli extract.
|
|
|
Deg1p Activity Is Sensitive to tRNA Three-dimensional
Structure--
Yeast tRNAPhe (anticodon GAA) is an
excellent experimental model to study the influence of overall
three-dimensional tRNA structure on the modification reaction. This
tRNA naturally contains 14 modified nucleosides, among them two
pseudouridine residues (at positions 39 and 55). The set of
tRNAPhe mutants with well defined disruptions in tertiary
interactions was also available (26). As shown in Fig. 6B
and Table II, almost 1.7 mol of /mol of tRNAPhe
transcript was formed upon the incubation with the yeast extract.
Intracellular Location of Deg1p--
The subcellular localization
of ProtA-tagged Deg1p in yeast cells was analyzed by indirect
immunofluorescence microscopy using tag-specific antibodies and by
direct fluorescence microscopy using a GFP-tagged version of Deg1p
which could also complement the deg1 strain.
In both cases a specific intranuclear signal could be detected, but the
cytoplasm was also diffusely stained (Fig.
7). These results show that Deg1p resides
in both the nucleus and the cytoplasm.
|
Pseudouridine Residues 38 and
39 Are
Absent in tRNA from Deg1
Strain--
The cellular
localization of Deg1p studied by immunofluorescence techniques reveals
the presence of nuclear and cytoplasmic pools of the protein but leaves
open the question about its presence in yeast mitochondria. To answer
this question we performed the analysis of pseudouridine residues
present (or absent) in tRNAs extracted from wild type and
DEG1-disrupted strain. Chemical mapping of pseudouridines
was done on total tRNA fraction by CMCT-reverse transcription
technique, as described previously (35). The synthetic oligonucleotides, complementary to the last 18-20 3
-nucleotides in
tRNA, were used for primer extension analysis using unmodified and
CMCT-treated tRNA. The results of reverse transcription for cytoplasmic
tRNAGly (anticodon GCC) and mitochondrial
tRNAArg (anticodon ACG) extracted from wild type and mutant
strains are presented in Fig. 8,
A and B. The strong reverse transcription stops
corresponding to
38 in cytoplasmic tRNAGly
and
39 in mitochondrial tRNAArg are totally
absent in the mutant DEG1-disrupted strain (indicated by
arrows). The formation of other pseudouridines naturally
present in these two tRNAs (
32 and
55 in
cytoplasmic and mitochondrial tRNA respectively) is not affected by
DEG1 gene disruption. These results confirm the absolute
requirement of Deg1 protein for modification of the pool of cytoplasmic
tRNAs and reveal that the product of the same gene DEG1
participates also in pseudouridine formation in mitochondrial tRNAs.
|
Deg1p Is Not Linked Genetically to Pus1p or Los1p--
We have
shown previously that Pus1p interacts genetically with the nuclear
pore-associated proteins Nsp1p and Los1p (9), suggesting that tRNA
modification may be linked to tRNA nuclear export. To test this
possibility for Deg1p, we combined the deg1 disruption with
the los1 or pus1 disruption by mating the
corresponding strains and performing tetrad analysis. The
double-disrupted haploid strains
los1/deg1
and
pus1
/deg1
were viable
and did not exhibit any further growth defect than the single-disrupted
deg1
strain (data not shown). Therefore, the
functional interaction with the nuclear pore complex appears to be
specific for Pus1p and does not occur in the case of Deg1p, suggesting
that modification only at particular sites is important for the tRNA
transport process. Furthermore, the viability of the
pus1
/deg1
strain
shows that yeast cells can tolerate the absence of
modification in
positions 27, 34, 36, 38, and 39. Thus, despite the different specificity of Pus1p and Pus3p there is no obvious synergism between them.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Yeast Protein Deg1 Is tRNA:38/39
Synthase--
Direct evidence that the yeast Deg1 protein is a
tRNA:pseudouridine synthase comes from two types of experiments: by
detecting the corresponding enzymatic activity in S100 extracts of a
transformed E. coli strain harboring the yeast
DEG1 gene and by measuring the enzymatic activity of a
ProtA-Deg1 fusion protein purified from a deg1
yeast strain. In both cases, the protein catalyzes in vitro
formation of pseudouridines at position 38 or 39 of several tRNA
transcripts. Additional evidence came also from identification of the
lacking pseudouridines at position 38 or 39 in cytoplasmic
tRNAGly and in mitochondrial tRNAArg present in
DEG1-disrupted yeast strain. As predicted previously (21),
Deg1p is indeed the yeast homolog of E. coli tRNA:
synthase I (PSU-I, also called truA, initially discovered as
hisT gene product). Therefore, after Pus1 and Pus2 (9), this
is the third tRNA:
synthase (PseudoUridine
Synthase, Pus3) so far characterized in yeast.
Yeast Pus3 and E. coli truA Display Different Substrate
Specificity--
Testing several tRNA substrates allowed us to reveal
subtle differences in substrate specificity between yeast Deg1p and
E. coli truAp. Indeed, although both enzymes display rather
good cross-reactivity toward uridine 39 in two different tRNA
transcripts (tRNAPhe and tRNAHis), the
situation for 38 and
40 formation was
different. E. coli truA modified U40 fairly well
in both E. coli tRNASer and yeast
tRNAAsp, whereas the yeast Deg1p did not modify
U40 at all in the same tRNA transcripts. This observation
fits well with the fact that U40 in naturally occurring
yeast tRNAAsp is not modified into
, but in E. coli all U40-containing tRNAs bear
40
(42). This absence of
40 is valid only for tRNAs from fungi and does not apply for tRNAs from higher eukaryotes, where few
cases of
40-containing tRNAs were found (42). However, the above observation for U/
40 relationship in yeast
tRNAs is valid for both yeast cytoplasmic and the mitochondrial tRNAs, as in cytoplasm, several naturally occurring yeast mitochondrial tRNAs
contain
38 or
39 but not
40. This is consistent with the fact that the same gene
product modifies all tRNA substrates in the different cellular
compartments in yeast (nucleus, cytoplasm, and mitochondria; see
below).
A Single Nuclear Gene DEG1 Provides the Enzyme for Three Cellular
Compartments--
Immunochemical studies demonstrate that Deg1 protein
is mostly located in the nucleus, but a significant part of it is also found in the yeast cytoplasm. Moreover, the analysis of the
pseudouridine tRNA modification pattern in the
DEG1-disrupted strain demonstrates clearly that a single
gene DEG1 is responsible for the enzymatic formation of
38/39 in both cytoplasmic and mitochondrial tRNAs.
Deg1p Belongs to a truA-like Family That Is Distinct from All Other
RNA: Synthases Sequenced So Far--
Comparison of the amino acid
sequences of 12 truA-like proteins identified so far from
different organisms reveals the presence of highly conserved residues
(shown in gray in Fig. 1) within six blocks that are common
to all proteins of the family. Interestingly, almost the
same signature is present in Pus1p and Pus2p but is not found in any
other protein of the whole protein data bank (SwissProt and GenBank).
Only the sequence GRTDXGVHXG (block II in Fig.
1), bearing a conserved aspartic acid residue (indicated by a
dot in Fig. 1), is similar to amino acid sequence
GXRDXXXG (also referred as block II
by Koonin (40)) present in all other RNA:pseudouridine synthases. As
already proposed by Koonin, this universally conserved aspartic acid
residue may be implicated in the enzymatic catalysis. Recent
cross-linking studies and site-directed mutagenesis performed on
E. coli tRNA pseudouridine synthase I (prokaryotic homolog
of Pus3) clearly demonstrate that Asp-60 residue is essential for
enzymatic activity (46). Successful crystallization and resolution of
the structure of E. coli tRNA pseudouridine synthase I have
also been reported (47), which should shed light on the catalytic
mechanism of the uridine isomerization.
Disruption of the DEG1 Gene Influences Yeast Cell Growth--
In
yeast, gene DEG1 was discovered incidentally by
transcriptional analysis of the centromere region of yeast chromosome
VI. This gene is located very close to the centromere and is expressed at only a low level. Its disruption, although not lethal, causes a
pronounced slow growth phenotype (DEpressed
Growth) (21). In this work, we show that this slow growth
phenotype, which is much more pronounced at 37 °C, is entirely the
result of the absence of Deg1 protein as it can be complemented by the
corresponding gene or by clones expressing N-terminally tagged
proteins. The absence of the Deg1p correlates with the absence of at positions 38 and 39 of tRNA.
![]() |
ACKNOWLEDGEMENTS |
---|
The plasmids containing various tRNA genes were kindly provided by Drs. O.Uhlenbeck (Boulder, CO-USA), R.Giegé and F.Fasiolo (IBMC, Strasbourg, France), J.Bell (Univ. of Alberta, Canada) and Z.Szweykowska-Kulinska (Univ. of Poznan, Poland). The plasmid carrying the PNop1-GFP cassette was kindly provided by K. Hellmuth. We thank Dr. J-P. Waller (CNRS, Gif-sur-Yvette) for critical reading of the manuscript and useful comments.
![]() |
FOOTNOTES |
---|
* This work was supported in part by research grants from CNRS and Actions de la Recherche sur le Cancer (to H. G.) and by Research Grant Hu363/6-2 from the Deutsche Forschungsgemeinschaft (to E. C. H.).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.
¶ Supported by the Medical Research Council of Canada Operating Grant MRC-MT-1226 awarded to Dr. B. Lane, University of Toronto, Canada. Correspondence should be addressed to Y. Motorin, Laboratoire d'Enzymologie et Biochimie Structurales, Avenue de la Terrasse, Bat. 34, CNRS, Gif-sur-Yvette, France. Tel.: 33-1-6982-3498; Fax: 33-1-6982-3129; E-mail: Yuri.Motorin{at}lebs.cnrs-gif.fr.
1
The abbreviations used are: , pseudouridine
(5-(
-D-ribofuranosyl)uracil; CMCT,
1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate; PCR, polymerase chain reaction;
ORF, open reading frame; GFP, green fluorescent protein; ProtA,
S. aureus protein A; Bicine,
N,N-bis(2-hydroxyethyl)glycine.
2 K. Hellmuth and E. C. Hurt, unpublished data.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|