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J. Biol. Chem., Vol. 275, Issue 36, 27681-27688, September 8, 2000
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,From The Skaggs Institute for Chemical Biology and Departments of Molecular Biology and Chemistry, The Scripps Research Institute, La Jolla, California 92037
Received for publication, April 21, 2000, and in revised form, June 20, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the yeast Saccharomyces
cerevisiae, two genes (GRS1 and GRS2)
encode glycyl-tRNA synthetase (GlyRS1 and GlyRS2, respectively). 59%
of the sequence of GlyRS2 is identical to that of GlyRS1. Others have
proposed that GRS1 and GRS2 encode the
cytoplasmic and mitochondrial enzymes, respectively. In this work, we
show that GRS1 encodes both functions, whereas
GRS2 is dispensable. In addition, both cytoplasmic and
mitochondrial phenotypes of the knockout allele of GRS1 in
S. cerevisiae are complemented by the expression of the
only known gene for glycyl-tRNA synthetase in Schizosaccharomyces
pombe. Thus, a single gene for glycyl-tRNA synthetase likely
encodes both cytoplasmic and mitochondrial activities in most or all
yeast. Phylogenetic analysis shows that GlyRS2 is a predecessor of all
yeast GlyRS homologues. Thus, GRS1 appears to be the result
of a duplication of GRS2, which itself is
pseudogene-like.
Aminoacyl-tRNA synthetases
(aaRSs)1 determine the
genetic code by matching amino acids to their cognate anticodon-bearing
tRNAs. The nature of the specificity of tRNA recognition by aaRSs is of
particular interest when a single aaRS aminoacylates distinct tRNA
isoacceptors. In this circumstance, the nucleotides that are common to
the isoacceptors contain the "identity determinants" needed for
specific aminoacylation, whereas those that are different are imagined
to be less significant for aminoacylation. The compartmentalization of
the protein translation apparatuses within the cytoplasm and organelles
(such as mitochondria) of eukaryotes results in tRNA isoacceptors that
are distinguished not only by their sequences but also by their spatial
locations. This situation commonly results in the need for two genes
encoding distinct proteins for the same aminoacylation activity, with
one protein localized to the cytoplasm and the other to the
mitochondria. Each enzyme is then specific for the isoaccepting tRNAs
within its respective cell compartment but need not charge the
isoacceptors outside of the compartment to which it is localized. In
contrast, we report here a situation wherein the yeast
Saccharomyces cerevisiae each of two genes encode a
glycyl-tRNA synthetase, but where, surprisingly, only one of these two
is needed for function in both the cytoplasmic and mitochondrial compartments.
The catalytic domains of tRNA synthetases have one of two distinct
architectures, designated class I and class II (1-6). Most commonly,
class I enzymes are monomeric while class II synthetases are
Whole genome sequencing of the yeast S. cerevisiae revealed
that it harbors two genes for GlyRS (17-19). These genes are
GRS1 on chromosome II and YPR081C (referred to here as
GRS2) on chromosome XVI. Both genes encode proteins similar
to the Cloning GRS1 and GRS2 from S. cerevisiae and the
Schizosaccharomyces pombe GRS1 Homologue--
Standard
Escherichia coli and yeast molecular biological techniques
(21, 22) were employed for all procedures described below. S. cerevisiae GRS1 and GRS2, as well as S. pombe SPAC3F10.03 (hereon referred to as GRS1 (23))
were cloned into plasmid YEplac181 (24). S. cerevisiae GRS1
and GRS2 were amplified by PCR from S. cerevisiae
strain EFW6 (25). The S. cerevisiae GRS1- and GRS2-bearing blunt-ended products were ligated into
SmaI-digested YEplac181, and clones were isolated for which
the open reading frame of the gene was in the same orientation as the
plasmid-borne lacZ gene, thus yielding pTSScII-comp
(GRS1) and pTSScI-comp (GRS2). A 2.6-kb fragment
containing S. pombe GRS1 (2.0 kb coding sequence plus 0.57 kb of upstream DNA and 0.11 kb of downstream DNA) was amplified from
strain KGY246 (a kind gift from Peter Orlean, University of Illinois at
Urbana-Champaign) with the appropriate phosphorylated oligonucleotides.
The product was cloned into pCR-Blunt (Invitrogen, Carlsbad, CA), and a
2.4-kb GRS1-bearing KpnI fragment was removed and
subcloned into KpnI-digested YEplac181. This yielded a
plasmid (pTSSp1-2) in which the open reading frame of S. pombe
GRS1 was in the same orientation as the plasmid-borne
lacZ gene.
The following plasmids were used for the expression of GlyRS1. Plasmid
pTSScII-maint was constructed by ligating the
GRS1-containing SphI-SacI fragment
from pTSScII-comp into similarly digested YCplac33 (24). Plasmid
pTSScII-6His-5 was constructed by replacing the KpnI
fragment with the appropriate phosphorylated and annealed oligonucleotides and placed in the correct orientation to encode a
C-terminal His6 tag of GlyRS1. Plasmid pTSScII-comp10e is
similar to pTSScII-comp, except that it encodes an N-terminal truncated GRS1 with a new start codon behind the ADH promoter from
pQB261 (Cubist Pharmaceuticals, Cambridge, MA). This promoter was
separated from the coding sequence by stop codons present in all three
reading frames.
The following plasmids were used for the expression of GlyRS2. Plasmid
pTSScI-6His was constructed from pTSScI-comp by replacing the normal
C-terminal coding region with one containing a His6-tag. Plasmid pTSScI-ADH6H is similar but was constructed by replacing the normal promoter with the ADH promoter.
Chromosomal Deletion and Analysis of S. cerevisiae GRS1 and
GRS2--
GRS1 and GRS2 were completely ablated
from the S. cerevisiae chromosome using the short flanking
homology-PCR method described by Baudin et al. (26).
Standard genetic techniques were employed (22).
The construction of the GRS1 null allele, RJT3/II-1 (MAT
The GRS2 null strain, RJTglys1-1 (MAT Isolation of mRNA and Relative Quantitation by
RT-PCR--
mRNA was isolated from S. cerevisiae FY250
cells that were grown to mid-logarithmic phase in defined synthetic
medium supplemented with 2% glucose as the carbon source, using the
Poly(A)Pure mRNA isolation kit (Ambion, Austin, TX). The cDNA
was then synthesized from the polyadenylated mRNA using a
RETROscript First-Strand synthesis kit for RT-PCR (Ambion).
Western Blot Analysis for Detection of Expression of GlyRS1 and
GlyRS2--
Protein samples were analyzed from S. cerevisiae FY250 cells that were grown to mid-logarithmic phase in
defined synthetic medium supplemented with 2% glucose as the carbon
source. The expression of His6-tagged proteins was detected
with the penta-His antibody (Qiagen, Valencia, CA) and was performed
using standard protocols (28).
Expression and Purification of His6-labeled GlyRS1
and GlyRS2--
GlyRS1 and GlyRS2 were purified from FY250 cells
bearing plasmids pTSSCII-6His-5 and pTSScI-ADH6H, respectively. Cells
were grown to mid-logarithmic phase in defined synthetic medium
supplemented with 2% glucose as a carbon source. Cells were harvested
and lysed by a French press. GlyRS1 and GlyRS2 were purified using
nickel-nitriloacetic acid agarose (Ni-NTA, Qiagen), eluting the
His6 proteins with an imidazole concentration gradient.
Aminoacylation Assays--
Aminoacylation assays were performed
at 37 °C using trichloroacetic acid precipitation. Incubation
mixtures contained 200 µg/ml bovine serum albumin, 100 mM KCl, 15 mM MgCl2, 20 mM dithiothreitol, 10 mM ATP, 50 mM
Hepes, pH 7.2, 5 µM [3H]glycine, 200 µM yeast total tRNA, and varying amounts of enzyme. No
RNA controls were used to correct for background.
S. cerevisiae GRS1 and GRS2 Encode Similar Proteins--
The
similarity of the sequences of S. cerevisiae GlyRS1 and
GlyRS2 is evident from their alignment (Fig.
1). In particular, the sequence of GlyRS2
(Sce2) is 59% identical and 87% similar to that of GlyRS1 (Sce1). The
only obvious difference between the two proteins is a 34-amino acid
insertion that is present in GlyRS1. This insertion is found within an
active site subdomain that is predicted to contact the acceptor stem of
the tRNA substrate (12). The known yeast GlyRSs from S. pombe (Fig. 1, Spom) and Candida albicans
(Fig. 1, Calb) also posses this insertion. The insertion is
characterized by the lysine-rich consensus sequence KKKRKKKVK and by a
high density of arginine, aspartate, and glutamate residues. These four
residues comprise 83% and 68% of the insert in C. albicans
GlyRS and S. cerevisiae GlyRS1, respectively.
GRS1 Encodes Both Cytoplasmic and Mitochondrial GlyRS
Activities--
Cytoplasmic aaRSs are essential proteins, so that
deletion of any one of their genes is typically lethal to the cell.
Others have inferred that GRS1 is essential for growth of
S. cerevisiae, because they were unable to isolate
kanamycin-resistant spores from a diploid strain that was heterozygous
for GRS1
(GRS1/grs1::KANR) (29). In the
present study, the essential nature of GRS1 was established
independently by a direct method. For this purpose, GRS1 was
ablated from the chromosome and replaced with the HIS3 marker. To maintain cell viability, a plasmid bearing GRS1
and URA3 (pTSScII-maint, see "Materials and Methods")
was introduced. The resultant strain was designated RJT3/II-1, and its
genotype was verified by PCR analysis (Fig.
2, A and B).
To check for the essential nature of GRS1, we took advantage
of the observation that retention of the URA3-bearing
plasmid is lethal when cells are grown in the presence of 5-FOA.
Lethality results because URA3 encodes
orotidine-5'-phosphate decarboxylase, which catalyzes a step in the
conversion of 5-FOA into the toxic nucleotide analogue
5-fluorodeoxy-UMP, an inhibitor of thymidylate synthase (30-32). Thus,
the URA3-bearing plasmid must be lost for cell survival.
However, loss of the plasmid also results in the loss of the sole copy
of GRS1 so that, if GRS1 is essential, growth on
5-FOA is lethal. In this case, growth can be rescued with an exogenous
source of the GRS1 gene product.
Growth of strain RJT3/II-1 in the presence of 5-FOA was observed when
the coding sequence of GRS1 and sufficient upstream DNA to
include all of its native expression signals were supplied on a
separate plasmid (Fig. 3 top,
GRS1). In contrast, RJT3/II-1 does not grow in the presence
of 5-FOA when GRS1 was absent from the plasmid (Fig. 3
top, Vector). Thus, GRS1 is essential
for providing cytoplasmic GlyRS activity in S. cerevisiae.
GRS2 was deleted directly from the chromosome of the haploid
strain FY250 to determine if it encoded mitochondrial GlyRS activity (see "Materials and Methods"). The genotype of the resultant strain (RJTglyS1-1) bearing the null allele was verified by PCR analysis (Fig.
2, A and B). Deletion of a mitochondrial aaRS is
lethal only when cells are grown on a non-fermentable carbon source
such a glycerol. Fig. 3 (middle) shows that deletion of
GRS2 was not lethal when the cells were grown in the
presence of either glucose (YPD) or glycerol (YPG). To confirm this
result, a heterozygous deletion of GRS2
(GRS2/grs2::HIS3) was constructed (in a
manner similar to that described under "Materials and Methods" for
the haploid strain). This diploid strain was sporulated, and a 4:0 (viable:nonviable) segregation of the tetrad was consistently observed
whether the carbon source was glucose or glycerol (data not shown).
Thus, GRS2 does not encode S. cerevisiae
mitochondrial GlyRS activity.
The lack of a phenotype for the GRS2 null allele raised the
possibility that GRS1 encodes mitochondrial as well as
cytoplasmic GlyRS activity. To examine mitochondrial GlyRS activity,
growth of 5-FOA-treated RJT3/II-1 cells was analyzed on YPG (glycerol) medium. Ectopic GRS1 supported growth on glycerol, showing
that there was no defect in mitochondrial GlyRS activity (Fig. 3,
bottom).
Because most mitochondrial targeting signals are located at the N
terminus, we tried to create a mitochondrial defect by deleting amino
acids 2-12 of GlyRS1. A deletion was created where codons 2-12 were
deleted and codon 13 was preceded by a new AUG start codon. The
resultant deletion protein was expressed from the ADH promoter and was
designated ADH/GRS1 The Sole Glycyl-tRNA Synthetase from S. pombe Fully Rescues the S. cerevisiae GRS1 Null Allele--
Although over 90% of the genome of
S. pombe has been sequenced, only a single gene encoding
GlyRS (GRS1) has been
revealed.2 As described
above, the protein product of this gene bears the same insert found in
GlyRS1 and not present in GlyRS2. Thus, GlyRS1 may be the source of
both cytoplasmic and mitochondrial GlyRS activity in most or all
yeasts. This possibility was examined further by determining whether
S. pombe GRS1 could complement the S. cerevisiae
GRS1 null allele. For this purpose, S. pombe GRS1 and
sufficient upstream DNA to include all of its expression signals was
cloned into YEplac181. The resultant plasmid (pTSSp1-2) was able to
support growth of the GRS1 null strain RJT3/II-1 on 5-FOA
(Fig. 3, top, SpGRS1), YPD, and YPG (Fig. 3,
bottom, SpGRS1). We surmise that a single GlyRS
can provide both cytoplasmic and mitochondrial GlyRS activity in
S. pombe.
Expression of GlyRS2 Fails to Rescue the GlyRS1-deficient
Strain--
Strikingly, GRS1 is essential despite the
presence of the homologous gene GRS2. We tested whether
GRS2 would be sufficient when present in multiple copies. To
pursue this question, GRS2 and sufficient upstream DNA to
include all of its native expression signals was cloned into plasmid
YEplac181. This plasmid contains the 2µ origin and, therefore,
should be present in 20-50 copies per cell. The resultant plasmid
(designated pTSScI-comp) was tested for its ability to rescue growth of
RJT3/II-1. This experiment showed that YEplac181-borne GRS2
was unable to compensate for the deletion of GRS1 (Fig. 3,
top).
To examine whether the lack of complementation of the GRS1
null allele by GRS2 was the result of poor expression, a tag
of 6 histidines (His6-tag) was placed onto the C terminus
of GlyRS2 (pTSScI-6His) and of GlyRS1 (pTSScII-6His-5). This tag
facilitated the determination of GlyRS1 and GlyRS2 expression by
Western blot analysis using the penta-His antibody (Qiagen). With this
system, GlyRS1 (GRS1-His6) was readily detected,
whereas GlyRS2 (GRS2-His6) was barely detectable
(Fig. 4). The native GRS2
promoter region was then replaced with the strong ADH promoter
(pTSScI-ADH6H), and this replacement resulted in detectable levels of
GlyRS2 (ADH/GRS2-His6), albeit at substantially
lower levels than GlyRS1. With GRS1 present in 20-50 copies
per cell instead of the normal 1 or 2 copies, the quantity of
chromosomally produced GlyRS1 was estimated by loading 20- and 40-fold
less of the GRS1-His6 sample (0.05× and 0.025×, respectively). Comparison of 0.05×- and
0.025×-GlyRS1-His6 to ADH/GlyRS2-His6
suggested that GlyRS2 expression was comparable to the level of
expression of GlyRS1 that would be expected from 1 or 2 copies of
GRS1. However, when these His6-tagged proteins were examined for their ability to complement the GlyRS1-deficient strain RJT3/II-1, only GlyRS1 (GRS1-His6) and
not GlyRS2 (GRS2-His6 and
ADH/GRS2-His6) resulted in growth on 5-FOA (Fig.
3, top). The ability of His6-tagged GlyRS1
(GRS1-His6) to complement both the cytoplasmic
and mitochondrial activity of the GlyRS1-deficient strain (Fig. 3,
top and bottom) demonstrates that the C-terminal His6-tag results in no loss of complementation activity and
likely does not account for the lack of complementation by
His6-tagged GlyRS2.
Chromosomal GRS2 mRNA Is Poorly Expressed--
The lack of
significant expression of S. cerevisiae GlyRS2 when
GRS2 was present in many copies per cell (Fig. 4,
GRS2-His6) prompted an examination of
GRS2 transcription from its wild-type promoter. The high
degree of identity between GRS1 and GRS2 mRNA sequences, coupled with the size difference resulting from the insertion sequence within GRS1, was exploited to determine
(by RT-PCR) the relative levels of GRS1 and GRS2
mRNA (Fig. 5A) in wild-type strain FY250. Polyadenylated mRNA was isolated from cultures grown to mid-logarithmic phase. GRS1- and
GRS2-specific cDNAs were synthesized using the
oligonucleotide RTPCR-R1, which annealed to the transcription products
of both genes (Fig. 5A, cDNA Synthesis 1). The relative
amounts of GRS1 and GRS2 mRNA were observed
by PCR amplification of the cDNA template using the
oligonucleotides RTPCR-F and RTPCR-R2, which flank the region of
GRS1 and GRS2 encoding the charge-rich insert
(Fig. 5A, PCR). The presence of the coding sequence for the
charge-rich insert within GRS1 yielded a product that was
almost 100 bp larger (434 bp) than the one produced from the
amplification of GRS2 (335 bp). Bias of the primer RTPCR-R1
toward synthesizing cDNA from GRS1 transcripts was
checked by synthesizing cDNA nonspecifically with the
oligonucleotide oligo-dT18 (Fig. 5A, cDNA
Synthesis 2). Contaminating chromosomal DNA, which would lead to
erroneous results, was detected by PCR amplification of mRNA to
which reverse transcriptase had not been added. Amplification could
only occur from contaminating genomic DNA template.
Amplification of the specific cDNA template (cDNA 1) derived
from FY250 mRNA (GRS1, GRS2) yielded the
expected 434- and 335-bp products (Fig. 5B). However, the
larger of the two bands (corresponding to GRS1) was
significantly more intense than the smaller band (GRS2). The
same amplification pattern was observed with the nonspecific cDNA
template (cDNA 2), demonstrating there wasn't bias in the cDNA
synthesis. Proclivity toward the amplification of one gene or the other
was examined by amplifying genomic DNA isolated from FY250 (genDNA),
where GRS1 and GRS2 were present in equimolar concentrations. The expected bands were observed but were present in
similar quantities. To verify that the smaller band resulted from
GRS2 amplification, the same oligonucleotides were used to amplify both cDNA and chromosomal DNA template from RJTglyS1-1 (GRS1, grs2::HIS3). In each
case, the smaller of the two bands was absent. No amplification was
observed from the mRNA template, demonstrating the probable lack of
chromosomal DNA contaminant within the cDNA preparation. Thus, the
concentration of GRS2 mRNA was very low relative to the
concentration of GRS1 mRNA.
GRS1 Is a Duplication of GRS2--
The high similarity of the
sequence of GRS2 to that of GRS1, and the lack of
expression of GRS2, provoked us to examine the possible
historical relationship between these and other genes for GlyRS. A
maximum parsimony analysis of
The protein sequences used for the full GlyRS tree shown in Fig.
6A were just the core active
site and anticodon-binding domains, which comprise only 60% of the
sequence of S. cerevisiae GlyRS2. With the limitation of the
included sequences possibly causing a distortion of the results,
another analysis was performed using only the yeast GlyRS sequences
rooted with the The presence of a second gene for glycyl-tRNA synthetase in
S. cerevisiae raised the possibility that S. cerevisiae had a distinct mitochondrial GlyRS activity. Although
individual genes encoding separate cytoplasmic and mitochondrial aaRS
activities is common for S. cerevisiae and most other
eukaryotes (37-51), this situation had not been demonstrated for
eukaryotic GlyRSs. The data presented here show that S. cerevisiae GlyRS1 encodes both mitochondrial and cytoplasmic
functions. Mitochondrial and cytoplasmic activities for
alanyl-3, histidyl-, and
valyl-tRNA synthetases in S. cerevisiae are also encoded by
a single gene (52, 53). In contrast to the situation with GlyRS,
however, a second gene encoding AlaRS, HisRS, or ValRS does not exist
in S. cerevisiae (18).
A single gene for both cytoplasmic and mitochondrial GlyRS activities
has been proposed for humans, A. thaliana4, and
Caenorhabditis elegans (11, 54, 55). For this study, it is
probable that GlyRS1 is the sole GlyRS activity in all yeasts. The
identification of a possible GlyRS1 homologue in Aspergillus nidulans5 suggests that
this situation is likely for other fungi as well.
Involvement of S. cerevisiae GlyRS1 in mRNA 3'-end
formation through a direct interaction with tRNA-like structures in the pre-mRNA has been proposed (29). Such a role would require the targeting of GlyRS1 to the nucleus. Nuclear localization of GlyRS1 might be anticipated, because facilitation of export of tRNA is believed to be a general function of all eukaryotic aaRSs (56-58). We
considered the possibility that the function of the
GRS2-encoded protein was to facilitate export of
tRNAGly and, in addition, that this role was in itself
not essential. However, the lack of expression of GlyRS2
(Fig. 4) would seem inconsistent with this kind of
specialized role.
To investigate further GlyRS2, we attempted to purify it to assess its
activity. A stable form of GlyRS2 was not obtained, in two independent
attempts. In contrast, using the same protocol, a stable GlyRS1 was
isolated whose activity could readily be detected.
Consequently, the multifaceted nature of GlyRS1 leaves open to question
the role of the protein encoded by GRS2. The low level of
transcription of GRS2 suggests that GlyRS2 function has been completely supplanted by GlyRS1. It is conceivable that GRS2
expression is induced under some unknown condition. However, whole
genome cDNA microarray analyses of S. cerevisiae have
failed to uncover any condition that yields significant enhancement of
GRS2 expression, despite the examination of a variety of
growth conditions and genetic backgrounds (summarized by Proteome,
Inc., Beverly, MA). Thus, GRS2 is
pseudogene-like.
The impetus for the adoption of GlyRS1 over GlyRS2 (its predecessor)
remains in question. However, the location of GlyRS1's charge-rich
insertion, the only prominent distinguishing feature between these two
proteins, raises one obvious possibility. The insertion is located
within an active site subdomain that is predicted to contact the
acceptor stem of the tRNA substrate (12). Enhanced affinity or altered
specificity for tRNA resulting from the acquisition of this insertion
seems likely. In particular, the single GlyRS has to recognize both the
cytoplasmic and mitochondrial glycine tRNAs. These tRNAs have sequence
differences in their acceptor stems at places known to be important for
tRNA recognition by bacterial GlyRSs (59-61). However, A. thaliana and C. elegans do not contain this insert, and
yet in these organisms a single GlyRS must also aminoacylate a
mitochondrial tRNAGly that differs significantly in
sequence from cytoplasmic tRNAsGly.
If the charge-rich insert in GlyRS1 has nonspecific RNA binding
properties, these properties could give GlyRS1 a selective advantage
over GlyRS2. In this connection, the N-terminal appended domain of
S. cerevisiae GlnRS can enhance formation of productive synthetase-tRNA complexes (62). This domain has been shown explicitly to have nonspecific RNA binding activity and can be substituted with a
non-homologous, nonspecific RNA-binding domain from another protein.
Further support for the importance of nonspecific RNA binding functions
in tRNA synthetases is seen in the E. coli isoleucine and
alanine enzymes (63-65). These examples suggest that GRS2
has been rendered irrelevant by the enhanced interaction of tRNA with GlyRS1 rather than by any deficit in its own activity. This situation could have been enhanced by significant GlyRS1-tRNAGly
co-evolution, such that GlyRS2 is no longer able to replace GlyRS1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 dimers. However, glycyl-tRNA synthetases (GlyRSs) have two distinct types of quaternary structures: (
)2
tetramers and 2 dimers (7-10). The known
()2-tetrameric GlyRSs are confined to bacteria and
plant chloroplasts, whereas the 2-dimeric GlyRSs are
distributed among all groups of organisms (7, 11-15). Even though the
catalytic domains of both types have the same class II-defining
architecture, their sizes and sequences bear little similarity to each
other. As a consequence, the two types are believed to have different
origins (16).
2-dimeric GlyRSs. Others proposed that
GRS1 and GRS2 encode distinct cytoplasmic and
mitochondrial activities, respectively (20). Although no genetic or
biochemical basis for this suggestion was given, distinct genes for
mitochondrial and cytoplasmic functions are known for many other tRNA
synthetases in S. cerevisiae. Because of our interest in
understanding the evolution and development of glycyl-tRNA synthetases
in eukaryotes, we set out to investigate the biological significance of
each gene for viability of S. cerevisiae under conditions of
growth that did and did not require mitochondrial function. We also
tried to understand the possible historical relationship of GlyRS1 to GlyRS2.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
grs1::HIS3,
his3
200, leu21,
lys2202, trp163,
ura3-52, pTSScII-maint (cen, GRS1,
URA3)), was modified in accordance with protocols used for
the deletion of other essential aminoacyl-tRNA synthetase genes
from S. cerevisiae (25, 27). Strain RJT1 (MATa/,
his3200/his3200, leu21/leu21,
LYS2/lys2202,
TRP1/trp163,
ura3-52/ura3-52) was created by mating the haploid strains
FY837 (MATa, his3200,
leu21, lys2202,
ura3-52) and FY250 (MAT,
his3200, leu21,
trp163, ura3-52), which were kind
gifts from Professor Leonard Gaurente (Massachusetts Institute of
Technology, Cambridge, MA). A diploid strain heterozygous for
GRS1 (RJTglyS3 (MATa/,
GRS1/grs1::HIS3, his3200/his3200,
leu21/leu21,
LYS2/lys2202,
TRP1/trp163, ura3-52/ura3-52)), was constructed by introducing the
HIS3 marker (flanked by GRS1 upstream and
downstream non-coding sequences) into the diploid strain RJT1. Double
recombinant His+ products were selected, and proper
integration of the HIS3 marker at the GRS1 locus
was determined by PCR (see "Results"). Plasmid pTSScII-maint
(GRS1, URA3, see above) was introduced into
RJTglyS3, and the diploid cells were sporulated and dissected.
His+ and Ura+ haploid cells were then selected,
to give strain RJT3/II-1. The structure of the GRS1 locus of
RJT3/II-1 was confirmed by PCR analysis.
,
grs2::HIS3,
his3200, leu21,
trp163, ura3-52), was constructed
directly from the haploid strain FY250 (see above). The HIS3
marker flanked by GRS2 upstream and downstream non-coding
sequences was generated similarly to that done for GRS1.
Double recombinant His+ products were selected, and proper
integration of the HIS3 marker at the GRS2 locus
was demonstrated by PCR analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of yeast GlyRSs. Yeast GlyRSs
were aligned using ClustalW 1.7. Sce1, Sce2,
Calb, and Spom designate S. cerevisiae
GlyRS1, S. cerevisiae GlyRS2, and the only known GlyRSs from
C. albicans and S. pombe, respectively. Identical
residues are shaded in black. Conserved residues are shaded
in gray.
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Fig. 2.
PCR analysis of GRS1 and
GRS2 null alleles. A, a schematic of
the PCR strategy used to discern the insertion of HIS3 at
the proper locus within the S. cerevisiae genome. The genes
were probed from both directions (5' and 3', relative to the direction
of transcription as indicated by the bent arrow).
Arrows indicate the position of hybidization of the
oligonucleotide to the chromosomal DNA. The arrowhead
indicates the 3'-end of the oligonucleotide. The oligonucleotides
external to the gene are specific for the locus (either GRS1
or GRS2), whereas the internal oligonucleotides are
gene-specific (GRS1, GRS2, or HIS3).
The internal oligonucleotides for GRS1 and GRS2
(2 and 3) hybidize to regions common to both genes. B,
results of the analysis of the GRS1 null allele (RJT3/II-1),
the wild-type precursor (RJT1), and the diploid intermediate
(GRS1/grs1::HIS3) in the
GRS1 null allele construction (RJTglys3, see "Materials
and Methods"), as well as the GRS2 null allele
(RJTglys1-1) and its wild-type precursor (FY250). The results clearly
show that all strains possess the proper genes at the proper
loci.
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Fig. 3.
Analysis of the growth of GRS1
and GRS2 null alleles on various media.
GRS1 encodes cytoplasmic GlyRS activity (top
panel). The GRS1 null allele (RJT3/II-1) was
transformed with various plasmids derived from the vector YEplac181
(Vector) expressing wild-type and C-terminal His6 fusions
of: GlyRS1 (GRS1, pTSScII-comp;
GRS1-His6, pTSScII-6His-5) and GlyRS2
(GRS2, pTSScI-comp; GRS2-His6,
pTSScI-6His) from their native promoters; GlyRS1 with a deletion of
residues 2 through 12 (ADH/GRS1 2-12, pTSScII-comp10e)
and C-terminal His6-tagged GlyRS2
(ADH/GRS2-His6, pTSScI-ADH6H), each expressed
from the ADH promoter; and the S. pombe GlyRS1 homologue
expressed from its native promoter (SpGRS1, pTSSp1-2).
Complementation was scored by the ability to grow in the absence of the
GRS1-containing maintenance plasmid (pTSScII-maint), which
is lost when the cells are grown in the presence of 0.1% 5-FOA.
GRS1 also encodes mitochondrial GlyRS activity (bottom
panel). Complementation is scored by the ability to grow in a
medium containing a non-fermentative carbon source, which requires
active mitochondria. Survivors of growth in the presence of 5-FOA were
streaked directly from the 5-FOA-containing medium (for which 0.2%
glucose was the carbon source) onto YPG (which contains 0.3%
glycerol). The constructs are the same as given above. GRS2
is dispensable (middle panel). The GRS2 null
allele (grs2, RJTglys1-1) and its wild-type
predecessor (FY250) were examined for growth on both fermentative (YPD,
0.2% glucose) and non-fermentative (YPG, 0.3% glycerol) carbon
sources.
2-12. Although the deletion protein
supported growth on glucose (5-FOA, Fig. 3, top; YPD, Fig.
3, bottom), it was unable to support growth when glycerol was the sole carbon source (Fig. 3, bottom). Thus, removal
of residues 2-12 of GlyRS1 selectively inactivated it for
mitochondrial but not cytoplasmic function. These results support the
conclusion that GlyRS1 provides both cytoplasmic and mitochondrial function.
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Fig. 4.
Analysis of the expression of GlyRS1 and
GlyRS2 by Western blot. Shown is a Western blot of FY250 bearing
plasmids derived from the vector YEplac181 (Vector) expressing
C-terminal 6-His fusions of GlyRS1 (GRS1-His6,
pTSScII-6His-5), as well as GlyRS2 from both its native and the ADH
promoters (GRS2-His6, pTSScI-6His;
ADH/GRS2-His6, pTSScI-ADH6H). The C-terminal
His6 fusion proteins were probed with the penta-His
antibody (Qiagen).
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Fig. 5.
Analysis of the expression of GRS1
and GRS2 by RT-PCR. A, schematic
of the RT-PCR experiment used to determine the relative level of
expression of GRS1 and GRS2 mRNA in the
GRS2 null allele (grs2, RJTglyS1-1) and the
wild-type strain from which it was derived (wt, FY250). See the
text for explanation. Oligonucleotides: RTPCR-F,
5'-CATGTWGACAAATTTWCTGATTGGATGTG-3'; RTPCR-R1,
5'-TCAGCACAWCCGACACATTCAATCC-3'; RTPCR-R2,
5'-AATTGWCCTTGKGCAGTYTCTGGCCT-3'. B, the products of RT-PCR
analyses of various templates separated on a 2% agarose gel.
2-dimeric GlyRSs from all
three of the major branches of life was conducted using the maximum
parsimony method (Protpars, part of the Phylip package of phylogenetic
programs (33)). Bias in the alignments of these proteins was eliminated
by reducing them to their core active site and anticodon-binding
domains. These portions comprised 83% of the smallest known GlyRS
(Mycoplasma genitalium). S. cerevisiae GlyRS1 and
GlyRS2 clustered within a monophyletic branch with the other known
yeast GlyRSs, with GRS1 arising from a duplication of
GRS2. The tree, including the relationship of the yeast
GlyRS1 homologues, was reasonably consistent with those based on
sequences of small-subunit rRNAs (34, 35) with the exception of the eukaryotes and the archae being paraphyletic, an observation also made
by Woese et al. (36). An analysis using the neighbor-joining method (Protdist, part of the Phylip package of phylogenetic programs (33)) also showed GRS1 arising from a duplication of
GRS2 (data not shown). The observation that the charge-rich
insert is, thus far, unique to the yeast GlyRS1 homologues and that no
other known GlyRS (except possibly the GlyRS from Plasmodium
falciparum) even possess an insert at this location provides
further support that GRS2 is ancestral to
GRS1.
2-GlyRS from Arabidopsis thaliana, because it was consistently located prior to the yeast node in our analyses. In this case, removal of all small extensions and
insertions that were not common to the entire set of protein sequences
left almost 95% of the sequence of GlyRS2. Both maximum parsimony and
neighbor-joining methods yielded the same basic tree (Fig.
6B) as that seen with the full set of GlyRS sequences (Fig. 6A), with S. cerevisiae GlyRS2
appearing as a predecessor of S. cerevisiae GlyRS1 and of
its homologues.
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Fig. 6.
Phylogenetic analysis of the relationship of
GlyRS1, GlyRS2, and other
2-dimeric GlyRSs. Sequences were
aligned with ClustalW 1.7 (66) and all major extensions and
insertions were removed. A, unrooted maximum parsimony tree
of 2-dimeric GlyRSs. The aligned sequences consisted of
the core of the active site and the anticodon-binding domains. The
numbers at the branches correspond to bootstrapping
frequencies calculated from 1000 trees. B, tree of the yeast
GlyRSs rooted with the 2-dimeric GlyRS from A. thaliana. The same tree was obtained from both maximum parsimony
and neighbor-joining methods. The numbers, X/Y, at the
branches correspond to bootstrapping frequencies calculated from 1000 trees using maximum parsimony and neighbor-joining methods,
respectively. A. pernix, Aeropyrum pernix
(BAA80640); A. thaliana, Arabidopsis thaliana
(O23627); A. fulgidus, Archaeoglobus fulgidus
(O29346); B. mori, Bombyx mori (Q04451); B. burgdorferi, Borrelia burgdorferi (O51344); C. elegans, Caenorhabditis elegans (Q10039); C. albicans, Candida albicans (Preliminary sequence data
was obtained from The Sanger Center web site); C. tepidum,
Chlorobium tepidum (*); C. acetobutylicum,
Clostridium acetobutylicum (AE001437); D. radiodurans, Deinococcus radiodurans (AAF11606);
H. sapiens, Homo sapiens (P41250);
M. thermoautotrophicum, Methanobacterium
thermoautotrophicum (O27874); M. jannaschii, Methanococcus
jannaschii (Q57681); M. avium, Mycobacterium
avium (*); M. leprae, Mycobacterium leprae
(L78812); M. tuberculosis, Mycobacterium
tuberculosis (O65932); M. genitalium, Mycoplasma
genitalium (P47493); M. pneumoniae, Mycoplasma
pneumoniae (P75425); P. falciparum, Plasmodium
falciparum (*); P. gingivalis, Porphyromonas
gingivalis (*); P. aerophilum, Pyrobaculum
aerophilum; P. abyssi, Pyrococcus abyssi
(CAB49474); P. furiosus, Pyrococcus furiosus
(Utah Genome Center, Dept. of Human Genetics, University of Utah);
P. horikoshii, Pyrococcus horikoshii (BAA30726);
S. cerevisiae 1, Saccharomyces cerevisiae GlyRS1
(P38088); S. cerevisiae 2, Saccharomyces
cerevisiae GlyRS2 (AAB68130); S. pombe,
Schizosaccharomyces pombe (CAA93301); S. aureus,
Staphylococcus aureus (Staphylococcus aureus
Genome Sequencing Project, and B. A. Roe, Yudong Qian, A. Dorman,
F. Z. Najar, S. Clifton, and J. Iandolo with funding from the NIH
and the Merck Genome Research Institute); S. coelicolor,
Streptomyces coelicolor (CAB69725); S. solfataricus, Sulfolobus solfataricus
(Sulfolobus genome project); T. thermophilus,
Thermus thermophilus (P56206); T. pallidum,
Treponema pallidum (O83678). The number contained
in parentheses after the organism name is the GenBank 228 accession
number of the GlyRS protein sequence from that organism. An
asterisk in the parentheses indicates that preliminary
sequence data was obtained from The Institute for Genomic Research web
site.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
|---|
* This work was supported in part by National Institutes of Health Grant GM23562 and by a fellowship from the National Foundation for Cancer Research.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.
American Cancer Society postdoctoral fellow (Grant PF-4300).
§ Predoctoral fellow of the National Science Foundation.
¶ To whom correspondence should be addressed: The Scripps Research Inst., 10550 N. Torrey Pines Rd., Mail Code BCC-379, La Jolla, CA 92037. Tel.: 858-784-8970; Fax: 858-784-8990; E-mail: schimmel@scripps.edu.
Published, JBC Papers in Press, June 28, 2000, DOI 10.1074/jbc.M003416200
2 V. Wood, personal communication, The Sanger Centre, Cambridge, England.
3 T. L. Ripmaster, C.-C. Wang, and P. Schimmel, unpublished.
4 A.-M. Duchene, personal communication, Institut de Biologie Moleculaire des Plantes, Strasbourg, France.
5 Aspergillus nidulans cDNA Sequencing Project, B. A. Roe, D. Kupfer, H. Zhu, J. Gray, S. Clifton, R. Prade and J. Dunlap; this project is supported by funds from the NSF-EPSCoR program.
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ABBREVIATIONS |
|---|
The abbreviations used are: aaRS, aminoacyl-tRNA synthetase; AlaRS, alanyl-tRNA synthetase; GlnRS, glutaminyl-tRNA synthetase; GlyRS, glycyl-tRNA synthetase; ADH, alcohol dehydrogenase; 5-FOA, 5-fluoroorotic acid; RT-PCR, reverse transcriptase-polymerase chain reaction; YPD, yeast extract/peptone/dextrose medium; YPG yeast extract/peptone/glycerol medium, kb, kilobase(s).
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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