Aminoacyl-tRNA synthetases are essential enzymes that catalyze
the esterification of an amino acid to its cognate tRNA with exquisite
specificity. The combination of biophysical, biochemical and genetic
techniques have significantly deepened our understanding of the
structure and function of these
enzymes(1, 2, 3) . The amino acid sequences
of seryl-tRNA synthetases from Escherichia coli(4) , Thermus thermophilus(5) , Bacillus subtilis(6) , Coxiella burnetii(7) , Saccharomyces cerevisiae(8) , Chinese hamster
(partial)(9) , and human (
)are known. According to
common structural motifs they are typical representatives of class II
synthetases(10) . The crystal structures of two prokaryotic
enzymes isolated from E. coli(11) and T.
thermophilus(12) are quite similar, as is their mode of
interaction with tRNA
. We have been working with yeast
SerRS, (
)which shows only moderate similarity (about 30%)
with prokaryotic seryl-tRNA synthetases on the level of the primary
structure(5) , but still recognizes bacterial tRNA
and can functionally substitute for the bacterial enzyme in
vivo(13) . The overexpression of the yeast SES1 gene in E. coli generated high amounts of functional
enzyme(13) , although with somewhat lower specific activity and
slightly different electrophoretic mobility (
)compared to
the enzyme isolated from yeast. This raises the possibility that the
yeast enzyme may not be modified or folded correctly in the bacterial
host. In this paper we describe the purification of yeast SerRS from an
overproducing strain of S. cerevisiae. The alignment of the
primary sequences of all SerRS proteins reveals that the enzymes from
yeast(8) , Chinese hamster(9) , and human
contain C-terminal extension between 20 and 48 amino acids long
not found in prokaryotic synthetases. We speculated whether this
peptide was important for maintaining the structure of the eukaryotic
enzymes or if it had another function. Thus, we deleted the part of the S. cerevisiae SES1 gene encoding the short C-terminal domain
and analyzed the expressed truncated protein.
MATERIALS AND METHODS
General
Procedures
[
C]Serine (180.5 Ci/mmol) and
[
H]ATP (40 Ci/mmol) were from Amersham Corp. YPD
medium contained 1% yeast extract, 2% peptone, and 2%
glucose(14) . Selection for yeast auxotrophic markers was done
in a medium of 0.67% nitrogen base and 2% glucose lacking amino acids,
supplemented as needed with adenine (20 µg/ml), uracil (20
µg/ml), and amino acids (20-30 µg/ml). Sporulation medium
contained 1% potassium acetate and amino acids as needed for
auxotrophic diploids. For induction of the GAL promoter,
glucose was replaced by galactose (2%) in all liquid media and agar
plates. Transformation of yeast was performed by the lithium acetate
procedure or by electroporation(14) .
Strains and Plasmids
These are described in Table 1. SES1 was used for various purposes, and thus
several different constructs were made. Plasmid pBRSES1
used for disrupting the SES1 gene (see scheme in Fig. 2) contained the genomic SES1 yeast DNA fragment
inserted in the opposite orientation compared to
pBRSES1(15) . For the construction of the centromeric
plasmid pUN70SES1, the SalI/BamHI fragment
of pBRSES1 was used. The truncation of the SES1 gene
was carried out (see below) in pVTU-103SES1, where the BamHI cassette containing SES1(13) was
inserted behind the ADH (alcohol dehydrogenase) promoter. The
resulting gene, lacking the 60-bp encoding amino acids 443-462 of
SerRS, was named SES1C20. For complementation of the null
allele strain, a 2.1-kb SphI fragment containing SES1 or SES1C20 behind the ADH promoter was cloned
into YEp351 (16) to generate YEp351SES1 and
YEp351SES1C20, respectively. For overexpression, the BamHI cassettes with SES1 or SES1C20 were
cloned behind the GAL promoter in pCJ11 (17) to
generate the plasmids pCJ11SES1 and pCJ11SES1C20,
respectively.
Figure 2:
Disruption of the chromosomal SES1 gene. The plasmid pBRSES1
contains the coding
region of the yeast SES1 gene (solid box) as well as
the 5`- and 3`-noncoding regions (shaded box and arrow). In order to remove the HindIII site in the
vector, plasmid was digested with SalI and religated. The
internal 0.4-kb HindIII fragment of SES1 was replaced
with the entire yeast LYS2 coding region (empty box),
and the disrupted gene SES1::LYS2 was introduced into
diploid yeast cells, as a BsmI/ClaI fragment, by
electroporation. The rearrangement of the chromosomal region containing SES1 was confirmed by Southern blot, shown at the lower
part of the figure. Genomic DNA was isolated from strain BR2727,
before and after replacement of the chromosomal SES1 gene with
the disrupted mutant allele (lanes 1 and 2,
respectively). After digestion with BamHI, fragments were
separated by gel electrophoresis, transferred to nitrocellulose, and
probed with radioactively labeled entire SES1 gene. The
position of the marker DNA fragments are shown on the left
margin.
SES1 Gene Disruption
The experimental strategy (18) is presented in Fig. 2. In order to eliminate a HindIII site in the vector, pBRSES1
was
digested with SalI and the larger fragment religated. The
0.4-kb HindIII fragment of the SES1 gene was excised
and replaced with a LYS2 cartridge(19) . This is a
4.5-kb HindIII fragment with the LYS2 structural gene
behind the CYC1 promoter. A 5-kb BsmI/ClaI
fragment, in which the LYS2 gene was flanked by 137 and 379 bp
of SES1 gene sequence, was then transformed by electroporation
into S. cerevisiae diploid strain BR2727. Lys
transformants were selected, sporulated, and tetrads dissected.
Total DNA was also isolated from Lys
transformants and
used for Southern blot analysis.
In Vitro Mutagenesis
The SES1 gene was
truncated by loop-out in vitro mutagenesis, with a 1:1 molar
ratio of phosphorylated oligonucleotide to U-containing single-stranded
pVTU-103SES1 DNA. The structure of the synthetic 37-mer was as
follows: 5`-CATTCATGATATGCTATATTTATAAAAATTCTGGTTC-3`. After
mutagenesis, DNA was transformed into E. coli strain DH5
and the plasmids with deleted SES1 were selected by colony
hybridization using the mutagenic oligonucleotide as a probe.
Purification of Wild-type and Mutant SerRS from the
Overproducing Strain
Yeast strains that overproduce full-length
SerRS and its truncated mutant SerRSC20 were generated by
transformation of S. cerevisiae S2088 by plasmids
pCJ11SES1 and pCJ11SES1C20, respectively. The
transformants were grown in glucose selective medium at 30 °C until
saturation, and than diluted 25-fold with galactose-containing medium
for promoter induction. The growth was continued until late exponential
phase (A
2.5). In a typical experiment,
the culture (1500 ml) was harvested by centrifugation (giving about 3 g
of cells), washed with water, and resuspended in cold lysis buffer (100
mM Tris-HCl, pH 7.5, 5 mM DTT, 0.1 mM EDTA,
and 0.2 mM PMSF) to a volume of 8 ml, followed by addition of
an equal volume of glass beads (0.5 mm diameter). The disruption of
cells was carried out in the bead beater, equipped with a cooling
jacket, by four 15-s bursts, separated by 30 s of cooling. The cell
breakage was over 90%. The beads were rinsed with the lysis buffer. The
lysate was clarified by centrifugation at 100,000
g for 90 min at 4 °C, giving about 10 ml extract with 10 mg of
protein/ml. SerRS was purified by chromatography on DEAE-cellulose
(DE52, Whatman) and phosphocellulose (P-11 Whatman) columns, as
described previously(20, 21) . Protein extract was
dialyzed against 20 mM potassium phosphate buffer, pH 7.2,
containing 10% glycerol, 3 mM DTT, 0.1 mM EDTA, and
0.2 mM PMSF and applied onto a DEAE-cellulose column (1.5
14 cm) equilibrated in the same buffer. The column was
developed with 60 ml of potassium phosphate gradient (20 mM,
pH 7.2 to 250 mM, pH 6.8) in the presence of 10% glycerol, 3
mM DTT, 0.1 mM EDTA, and 0.2 mM PMSF.
SerRS-containing fractions were detected by aminoacylation assay and
SDS-PAGE, while in the case of SerRSC20 fractionation, the distribution
of full-length and truncated SerRS in the fractions eluted from
DEAE-cellulose column were detected by Western blots, using rabbit
antiserum raised against wild-type SerRS (8) . The proteins in
the pooled fractions were carefully desalted on a Sephadex G-25 column
(PD-10, Pharmacia Biotech Inc.) and applied to a phosphocellulose
column (1.6
5 cm) equilibrated in HEPES/KOH buffer, pH 7.0,
containing 10 mM potassium chloride, 10% glycerol, 3 mM DTT, 0.1 mM EDTA, and 0.2 mM PMSF. The column
was eluted with a 60-ml gradient of potassium phosphate (10-700
mM) in a buffer of the same composition as for the
equilibration of the column.
Determination of Kinetic Parameters
The
aminoacylation was done at 30 °C. Standard reaction mixtures
contained 50 mM Tris-HCl, pH 7.5, 15 mM MgCl
, 5 mM ATP, 4 mM DTT, 4 mg/ml
unfractionated brewer's yeast tRNA (Boehringer Mannheim), and 25
µM [
C]serine as
described(13) , except that the serine concentration was
increased up to 1 mM in selected experiments. Kinetic
parameters (k
and K
) of the
aminoacylation reaction were determined with unfractionated yeast tRNA
preparations containing 6% serine-accepting tRNAs(13) . The
concentrations of serine-accepting tRNA ranged from 30 to 1000 nM in the reactions with wild-type SerRS and from 10 to 270 nM with SerRSC20. The K
values for ATP and
serine were determined in the aminoacylation reaction by varying the
concentration of studied substrate: ATP from 2 to 150 µM and [
C]serine from 2 to 360
µM, respectively, while the other substrates were kept
saturating. The concentrations of SerRS and SerRSC20 were 2-5
nM. The SerRS active site concentration was determined by the
formation of the enzyme bound intermediate seryl-AMP according to Borel et al.(22) .
Southern and Northern Blot Hybridizations
Total
yeast DNA was isolated from transformed and non-transformed yeast
cells(14) . After digestion with BamHI, the fragments
were separated on 0.7% agarose gel, transferred to nitrocellulose
membrane and probed with 1.4-kb BamHI fragment (Table 1)
containing the entire SES1 coding region. For Northern
analysis, total yeast RNA was isolated by the guanidinium thiocyanate
method(14) , separated on denaturing formaldehyde gel,
transferred to Nytran membrane (Schleicher & Schuell), and
hybridized to the entire SES1 gene, as described in the
manufacturer's protocol.
RESULTS
Rationale
We wanted to test the functional and
structural importance of the C-terminal extension of yeast SerRS,
comprising 20 mostly basic and hydrophilic amino acids. Since this
peptide is not a part of three highly conserved motifs characterizing
class II synthetases(23) , and is not present in four
prokaryotic SerRS enzymes (5, 7, 6, 24) (Fig. 1), a
non-catalytic role was proposed. We therefore investigated charging by
the truncated protein, both in vivo and in vitro, as
well as its stability.
Figure 1:
Schematic
representation of the homology on the amino acid level between SerRS
from E. coli, T. thermophilus, B. subtilis, C.
burnetii, S. cerevisiae, Chinese hamster, and human. Numbers above the line indicate the position of the
amino acids. The composition of the C-terminal extensions in the yeast
enzyme (443-462) and the corresponding sequences of Chinese
hamster and human SerRS (469-493) are shown by one-letter
code. Only the sequence of 199 C-terminal amino acids of Chinese
hamster SerRS is known. Horizontal bars indicate the
approximate position of three motifs characteristic for class II
synthetases. There is extensive homology between all SerRS proteins in
motifs I, II, and III.
C-terminal Truncation of SerRS
A 60-bp fragment of
the SES1 gene, coding for 20 C-terminal amino acids of SerRS,
was deleted by in vitro loop-out mutagenesis using a
discontinuously homologous oligonucleotide. The truncated protein
SerRSC20 ends with Leu-442. It was expressed from a truncated SES1 gene, which has its natural TAA stop codon, followed by 19-bp of SES1 3`-flanking region up to the introduced BamHI
site (see ``Materials and Methods''). The removal of the
unusual, positively charged C-terminal peptide (amino acids
443-462; pI = 10.8), substantially changes the ionic
properties of the whole enzyme: the calculated pI is 5.6 for the
full-length protein and 5.3 for the truncated form.
Construction of S. cerevisiae SES1 Null Allele
Strain
In order to test activity of the SES1 truncation
mutant in vivo, we constructed the S. cerevisiae strain with inactivated SES1 allele, BR2727
SES1 (Fig. 2). As previously shown by Southern blot
analysis(8) , SES1 is a single-copy gene in the
haploid genome, residing on a 15-kb BamHI fragment of
chromosomal DNA. The SES1 gene contains an internal 0.4-kb HindIII fragment, which was replaced on the plasmid with a LYS2 cartridge, which also serves as a selectable marker.
Lys
transformants were selected, and the correct LYS2 integration at the SES1 locus was verified by
Southern blot analysis (Fig. 2). Total DNA from
BR2727
SES1 and the parental strain BR2727 was digested
with BamHI, separated by agarose gel electrophoresis, and
transferred to nitrocellulose membrane. Hybridization with a
radioactively labeled 1.4-kb BamHI fragment containing the
entire SES1 structural gene, revealed the presence of one
wild-type SES1 allele, giving a 15-kb BamHI fragment (Fig. 2, lane 1) of genomic DNA, while the other SES1 allele has been replaced in BR2727
SES1 by
the mutant allele SES1:LYS2 (Fig. 2, lane 2).
Since the LYS2 gene carries an internal BamHI site,
its integration into SES1 gives rise to two new BamHI
fragments of 14 and 5 kb. Seven Lys
isolates were
sporulated and their tetrads dissected. All viable spores were lysine
auxotrophs, and in no case did more than two of four separated spores
form colonies. Inactivation of SES1 renders the cell inviable,
indicating that the SES1 gene is essential and therefore
indispensable for cell growth. The deletion could be rescued when SES1 was supplied on a centromeric or multicopy plasmid.
Transformed diploids were sporulated and viable Lys
haploids were isolated by the method of Rockmill et
al.(25) . Selected haploids with a disrupted SES1 gene (Lys
phenotype) where SerRS function was
provided in trans (from a centromeric plasmid carrying
wild-type SES1 gene), were used for complementation
experiments via plasmid shuffling.
The C-terminal Extension Is Dispensable for Cell
Viability
The recipient S. cerevisiae haploid
BR2727
SES1, where viability of the cell is ensured by a
wild-type SES1 on the URA3-containing plasmid
pUN70SES1, was cotransformed with YEp351SES1C20,
which carries the LEU2-selectable marker and SES1C20
behind the ADH promoter. The function of the deleted gene was
then assayed by complementation of SerRS function, by growing double
transformants in the presence of uracil and replica plating the
Lys
Leu
colonies to 5-fluoroorotic
acid containing medium(14) . This excludes the presence of the URA3-containing plasmid with the wild-type S. cerevisiae
SES1 gene. The ability to select viable haploids carrying SES1C20, as the only source of seryl-tRNA synthetase activity,
shows that 20 amino acids(443-462) at the C terminus of the
enzyme are not required for the survival of the yeast cell, although
the growth rate was significantly impaired (doubling time increased by
70%) when compared to BR2727
SES1 complemented with the
same plasmid carrying wild-type SES1.
Overexpression and Purification of Wild-type and
Truncated Seryl-tRNA Synthetases from Yeast
In order to study
the effect of removal of the C-terminal extension on the activity and
the stability of the enzyme, full-length SerRS and mutant SerRSC20 were
purified from yeast. To facilitate the purification of substantial
amounts of proteins required for in vitro studies, we were
interested in developing a high level overexpression system. Only
4-fold overproduction of SerRS was determined by Western blot analysis
and aminoacylation with crude protein extract prepared from yeast cells
transformed with pVTU103SES1 (Table 2). The expression
of the truncated protein from pVTU103SES1C20 was negligible
under the same experimental conditions. Induction of the GAL promoter
on pCJ11SES1, transformed in strain S2088, resulted in
150-fold overproduction of full-length SerRS protein and a
corresponding increase of the serylation activity measured in the crude
protein extract (Fig. 3A, lanes a and b; Table 2). We found this system very suitable for the
purification of the wild-type enzyme. However, in the protein extract
made from induced yeast cells transformed with pCJ11SES1C20,
the overproduction of truncated protein, as judged by scanning of the
bands on the Western blot (not shown), was only 10 times above the
control (extract made from non-induced cells) (Table 2) and thus
undetectable on a Coomassie-stained gel (Fig. 3A, lanes c and d). In addition, the serylation activity
of the extract, determined by standard aminoacylation assay (25
µM serine) was very low. It seems, therefore, that the
C-terminal fragment of yeast SerRS influences the expression of the
protein or its accumulation in the cell. This poor level of
overexpression of the truncated protein, together with the necessity to
separate the enzyme from endogenous wild-type SerRS present in the
yeast strain S2088, made the purification procedure much more difficult
and inefficient. pCJ11SES1C20 transformants of
BR2727
SES1 could not be used as a source of truncated
protein, because the strain carries a mutation in the galactose
permease gene and is not fully galactose inducible(26) .
Figure 3:
The overexpression and purification of
SerRS and SerRSC20 from yeast. A, an equal amount of protein
extract (50 µg), prepared from cells transformed with the indicated
plasmids under inducing and non-inducing conditions, was separated by
SDS/8% PAGE and stained with Coomassie Brilliant Blue G250. Lane
a, pCJ11SES1, non-induced; lane b,
pCJ11SES1, induced; lane c, pCJ11SES1C20,
non-induced; lane d, pCJ11SES1C20, induced. Lanes
e and f contain full-length SerRS after purification on
DEAE-cellulose and phosphocellulose columns (2 and 4 µg,
respectively). B, chromosomally encoded SerRS and overproduced
SerRSC20 were separated on a DE52 column. Aliquots (40 µl) from the
eluted fractions were run on an SDS gel, and the proteins were
electrophoretically transferred to nitrocellulose and probed with
polyclonal antibodies against yeast SerRS(8) . The presence of
the bound antibody was detected by anti-rabbit antibody conjugated with
horseradish peroxidase. Lanes a and b, 100,000
g supernatant from S2088/pCJ11SES1C20 (70 and
15 µg of protein, respectively); lanes c-m, DE52
fractions; lane n, pure SerRS, 0.2 µg. C, the
separation of SerRSC20 on the P-11 column was followed by SDS-PAGE and
silver staining of the gel. Lane a, DE52 fraction (same as k on panel B) loaded on the P-11 column; lanes
b-d, three successive P-11 fractions with highest serylation
activity. Black arrow indicates the position of full-length
yeast SerRS; white arrow indicates the position of truncated
protein. The position of the marker proteins are shown in the left
margin (fructose-6-phosphate kinase, 84 kDa; pyruvate kinase, 58
kDa; fumarase, 48.5 kDa; lactate dehydrogenase, 36.5
kDa).
Full-length SerRS was purified to apparent homogeneity (Fig. 3A, lanes e and f) by a quick
two-step chromatographic procedure that involves fractionation on
DEAE-cellulose and phosphocellulose columns (20, 21) as the important steps in isolation of SerRS
from non-overproducing yeast strains. The procedure allows the
purification of 1 mg of protein from 1 g of yeast cells, with a
specific activity of 115 nmol mg
min
, as determined in the standard
aminoacylation assay with 25 µM serine. By increasing the
concentration of serine to 1 mM (see below), a specific
activity for the wild-type enzyme of 280 nmol mg
min
was obtained. The purified enzyme has two
active sites for seryl-AMP per protein dimer (i.e. full site
reactivity), as determined by the formation of the SerRS-bound
intermediate seryl-AMP according to the method of Borel et
al.(22) . The purification procedure for the truncated
SerRS mutant was essentially the same as for SerRS, except the overall
yield was 2 orders of magnitude lower (about 8 µg of protein/g of
cells), both due to lower expression and to the narrow pools taken from
both ion exchangers (see below). The separation of truncated enzyme
from the wild-type SerRS by fractionation on DEAE-cellulose column was
analyzed by the aminoacylation assay and by SDS-PAGE followed by
Western blot analysis (Fig. 3B). The activity peak was
found in fractions j and k. Since the best separation of
full-length and truncated SerRS enzymes was found in fraction k,
this material was applied on the phosphocellulose column after careful
desalting. Truncated protein was eluted with 10 mM KCl,
immediately following the majority of unbound proteins, indicating very
weak interaction of SerRSC20 with the anionic resin. Fig. 3C shows the separation of the proteins from the P-11 eluted
fractions on a silver-stained SDS gel. Apparently homogeneous truncated
enzyme eluted in fraction d, was concentrated and used in
additional experiments. When assayed in the presence of elevated serine
concentration (1 mM), SerRSC20 shows specific activity similar
to that of the full-length SerRS (257 nmol mg
min
).
Deletion of the C-terminal Peptide Affects the Kinetics
of Serylation and Decreases Thermal Stability of the
Protein
Kinetic parameters for wild-type SerRS and mutant
SerRSC20 were determined in the aminoacylation reaction (Table 3). Due to the lack of pure yeast tRNA
, the
experiments were performed with bulk yeast tRNA, 6% of which was
tRNA
, thus only relative K
values
for tRNA are meaningful. Mutant enzyme shows 3.6-fold higher affinity
for tRNA
at 30 °C. Interestingly, this increased
affinity is not detectable at 37 °C, which is probably the result
of a subtle, temperature-dependent, conformational change of the mutant
enzyme (see below). Another difference in the kinetic parameters
between SerRS and SerRSC20 is a 4-fold increased K
value for serine obtained for the mutant enzyme. There are no
significant differences in k
values for the
substrates, with mutant and wild-type enzyme, when measured at high
serine concentration (1 mM). It should be noted that our
experiments revealed a higher K
value for serine
for wild-type SerRS (Table 3) than reported earlier (10
µM)(27) , which was the reason for the increase in
[
C]serine concentration in the aminoacylation
assay. The observed differences in the kinetic parameters for
aminoacylation may be the consequence of the overall change in the
enzyme conformation due to deletion of the positively charged
C-terminal fragment, which can interact with other parts of the protein
via electrostatic interactions. The involvement of the C-terminal
peptide in the stabilization of the overall enzyme structure was
further demonstrated by the comparison of the thermal stability of the
full-length SerRS and the mutant SerRSC20. The proteins were first
incubated at 42 °C. After various time intervals, aliquots were
withdrawn and tested for aminoacylation activity. Much faster heat
inactivation of truncated protein compared to the full-length SerRS was
observed (Fig. 4), without significant degradation of the
protein, as confirmed by Western blotting (not shown). This confirms
that removal of the C-terminal fragment of SerRS strongly influences
protein stability. The truncated protein may be folded into a less
stable conformation and is therefore more accessible to the
intracellular proteolytic apparatus, which would then not allow high
level accumulation of SerRSC20 in the cell.
Figure 4:
Heat
inactivation curve of full-length (
) and truncated (
)
SerRS. An equal amount of protein was incubated at 42 °C in 50
mM Tris-HCl, pH 7.5, 2 mM DTT, 5% glycerol. At
various time intervals, aliquots were withdrawn and used in the
aminoacylation assay. The remaining serylation activity is shown; 100%
activity corresponds to 31 and 29 pmol of charged tRNA for full-length
and truncated enzyme, respectively. The concentration of serine in the
assay was 1 mM.
C-terminal Truncation of SerRS Does Not Affect Its mRNA
Level
This low level of overproduction of truncated SerRSC20
protein, raised the question of whether the transcriptional level of SES1C20 and the stability of its mRNA are altered. Thus, total
RNA was isolated from yeast strain BR2727 transformed with either
plasmid pVTU-103SES1 or pVTU-103SES1C20, which carry
the structural gene SES1 and its truncated form, respectively,
distal to the ADH promoter. Northern blot analysis showed that the
truncation of 60 bp at the 3`-end of the SES1 coding region,
does not influence mRNA production and/or its stability (Fig. 5). The multiple bands in lanes b and c (Fig. 5) may reflect the difference in the size of the
transcripts originated from chromosomally encoded and plasmid-contained SES1 genes.
Figure 5:
Northern blot analysis of total yeast RNA
isolated from strain BR2727 transformed with pVTU-103 (a, 50
µg), pVTU-103SES1 (b, 25 µg), or
pVTU-103SES1C20 (c, 25 µg). RNA was fractionated
on a 1.5% agarose/formaldehyde gel and transferred to a Nytran membrane
(Schleicher & Schuell). Hybridization to a 1.4-kb BamHI
fragment of
P-labeled yeast DNA, containing the SES1 gene, was at 42 °C, as was the final wash of the
membrane.
DISCUSSION
Alignments of primary structures of a number of
aminoacyl-tRNA synthetases have established that subunit sizes of
eukaryotic enzymes are often larger than those of the corresponding
enzymes from prokaryotic sources(28, 29) . These
polypeptide chain extensions are found to be located at one extremity
of the molecules, usually the N terminus, rather than as insertions
within the conserved regions. The exceptions are eukaryotic isoleucyl- (30, 31) and seryl-tRNA synthetases, which carry the
extensions at the C terminus. The common feature of N-terminal
extensions of eukaryotic synthetases, is a high content of basic amino
acids. However, there is no homology or common structural motif between
these fragments. As regards the functional significance of N-terminal
extensions, it was proposed earlier that this domain confers on the
synthetase the ability to bind to polyanionic carriers, promoting the
compartmentalization of these enzymes within the cytoplasm, through
electrostatic interactions with as yet unidentified cellular components
carrying negative charges(28, 32) . This hypothesis
was experimentally tested on the aspartyl (33, 34, 35) ,
glutaminyl(36, 37) , lysyl(28, 38) ,
and methionyl (39) systems. It was demonstrated that truncated
enzymes lose affinity for anionic
carriers(28, 33, 37) . Although in some cases
the extension, or part of it, was important for structure/activity of
the enzyme, extensions were not found to be essential for the viability
of the cells(36, 39) . These studies reinforce the
concept that eukaryotic synthetases have quasi-independent domains not
found in their prokaryotic counterparts, which may confer a function
distinct from aminoacylation. Recent experiments show that the
N-terminal extensions of mammalian aspartyl- and arginyl-tRNA
synthetases are responsible for mediating its association with the
multisynthetase complex(40, 41) , as also shown
earlier for rat liver arginyl-tRNA synthetase(42) .
In the
serine system, three eukaryotic seryl-tRNA synthetases, from S.
cerevisiae(8) , Chinese hamster(9) , and
human,
share sequence homology with their prokaryotic
counterparts from E. coli(4) , T. thermophilus(5) , C. burnetii(7) , and B.
subtilis(6) , except that they contain a C-terminal
fragment of 20 or more mostly basic and hydrophilic amino acids (Fig. 1). Interestingly, the sequence of the first 25
residues(469-493) in the C-terminal extension in human SerRS is
almost identical (with the exception of one amino acid) to the sequence
of the corresponding peptide of Chinese hamster enzyme.
Crystallographic studies have shown that two prokaryotic enzymes,
isolated from E. coli and T. thermophilus fold to
very similar conformations(5) . Since none of the eukaryotic
seryl-tRNA synthetases has been crystallized yet, the influence of the
C-terminal domain on the overall structure of the protein is unknown.
Although the deletion of the C-terminal extension of the yeast enzyme
is not essential for the viability of the cell, thermal stability of
the truncated enzyme determined in vitro is much below that of
the full-length form. The abundance of positively charged (lysines) and
hydrophilic (serines) amino acids in the C-terminal peptides of
eukaryotic enzymes (Fig. 1), makes it likely that this domain is
involved in maintaining the overall structure of the enzyme via
electrostatic interactions. While the truncated enzyme shows full
activity in the aminoacylation reaction in the presence of elevated
serine concentration, as shown by very similar specific activities and k
values for SerRS and SerRSC20 (Table 3),
its affinity for the substrates differs from the full-length protein.
At 30 °C, the K
for tRNA is 3.6-fold reduced
for truncated enzyme, while the K
for serine is
4-fold elevated. These relatively small differences in kinetic
parameters could be explained by an overall change of protein
conformation, rather than by the loss of direct contacts with substrate
binding sites, which would be expected to lead to far more dramatic
changes.