From the § Department of Chemistry, University of
Minnesota, Minneapolis, Minnesota 55455, the ** Department of
Biochemistry and Molecular Pharmacology, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107, and the Department
of Cell Biology, Cancer Institute, Japanese Foundation for Cancer
Research, Kami-Ikebukuro, Toshima-Ku, Tokyo 170, Japan
Received for publication, January 17, 2001, and in revised form, February 21, 2001
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ABSTRACT |
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Analysis of prolyl-tRNA synthetase (ProRS) across
all three taxonomic domains (Eubacteria, Eucarya, and Archaea) reveals
that the sequences are divided into two distinct groups. Recent studies show that Escherichia coli ProRS, a member of the
"prokaryotic-like" group, recognizes specific tRNA bases at both
the acceptor and anticodon ends, whereas human ProRS, a member of the
"eukaryotic-like" group, recognizes nucleotide bases primarily in
the anticodon. The archaeal Methanococcus jannaschii ProRS
is a member of the eukaryotic-like group, although its
tRNAPro possesses prokaryotic features in the acceptor
stem. We show here that, in some respects, recognition of
tRNAPro by M. jannaschii ProRS parallels that
of human, with a strong emphasis on the anticodon and only weak
recognition of the acceptor stem. However, our data also indicate
differences in the details of the anticodon recognition between these
two eukaryotic-like synthetases. Although the human enzyme places a
stronger emphasis on G35, the M. jannaschii enzyme places a
stronger emphasis on G36, a feature that is shared by E. coli ProRS. These results, interpreted in the context of an
extensive sequence alignment, provide evidence of divergent adaptation
by M. jannaschii ProRS; recognition of the tRNA acceptor
end is eukaryotic-like, whereas the details of the anticodon
recognition are prokaryotic-like. This divergence may be a reflection
of the unusual dual function of this enzyme, which catalyzes specific
aminoacylation with proline as well as with cysteine.
Recognition of tRNA by aminoacyl-tRNA synthetases provides the
basis for decoding genetic information. Through this recognition, each
of the 20 families of tRNA is specifically charged with an amino acid
by the cognate synthetase, so that the amino acid/anticodon trinucleotide relationship can be used to translate nucleic acid sequences into proteins. Although each tRNA molecule consists of ~76
nucleotides, only a few are important for specific aminoacylation, and
these are generally located in the acceptor end and in the anticodon
(1). In some cases, the position and identity of a strong recognition
element has been maintained through evolution (2-5), whereas there are
other examples where specificity determinants have changed to adapt to
alterations in the synthetase sequence or structure (6-12). For the
latter cases, tRNA-synthetase co-adaptation is necessary to preserve
the specificity of recognition and the fidelity of the decoding system
through evolution.
In the case of tRNAPro recognition by prolyl-tRNA
synthetase (ProRS),1 the
determinants for the eubacterial Escherichia coli enzyme are
located at both the acceptor end and in the anticodon sequence (6). In
contrast, those for the human enzyme are located only in the anticodon
(13). This shift of determinants reflects a change in enzyme sequence
and structure from E. coli to human. Indeed, based on the
crystal structure of ProRS of Thermus thermophilus and an
extensive sequence alignment of ProRS enzymes from all three taxonomic
domains, the E. coli and human synthetases are clearly
members of two distinct groups of ProRS (Fig. 1) (13-15). Although
both the "prokaryotic-like" and "eukaryotic-like" groups contain three conserved motifs that are characteristic of the class II
aminoacyl-tRNA synthetases, specific amino acid sequences that
constitute these motifs differ between the two groups. This difference
is particularly striking in the variable loop connecting the
To complement previous studies of E. coli and human ProRS
and to determine the relatedness of tRNA recognition by ProRS from all
three domains of life, we investigated here ProRS of the archaeon Methanococcus jannaschii. Although this ProRS belongs to the
eukaryotic group, its tRNA shares some acceptor stem features with
E. coli tRNAPro. In particular, M. jannaschii tRNAPro contains an A73 discriminator base,
which plays an important role in aminoacylation by E. coli
ProRS (6). In contrast, human tRNAPro contains a C73, which
is not a critical determinant for aminoacylation by the human enzyme
(13). The presence of A73 in M. jannaschii tRNAPro raises the question of whether tRNA recognition by
the archaeal ProRS might resemble that of the E. coli enzyme
by placing a strong emphasis on acceptor stem recognition. On the other
hand, the sequence similarity between the M. jannaschii and
human synthetases (Fig. 1) (13, 21) suggests that acceptor stem
recognition might follow that of the human enzyme, which does not
recognize position 73 in a base-specific manner.
In addition to aminoacylation of the cognate tRNAPro,
M. jannaschii ProRS has the novel ability to also catalyze
aminoacylation of M. jannaschii tRNACys with
cysteine (22, 23). This dual functional ProRS is unique to M. jannaschii, its close relatives Methanococcus
maripaludis and Methanobacterium thermoautrophicum
(22), and to the deep-rooted eukaryote Giardia lamblia (21).
The specific aminoacylation with both proline and cysteine is expected
to make unusual demands on the enzyme structure that might include
relocation of determinants required for specific aminoacylation of
M. jannaschii tRNAPro compared with those
important for the E. coli or human enzymes, which lack this
dual function. Here, the results of biochemical studies of
tRNAPro recognition by M. jannaschii ProRS,
together with an extensive sequence analysis, have provided new
insights into these questions.
Preparation of M. jannaschii tRNAPro--
The
wild-type gene for M. jannaschii tRNAPro was
constructed by oligonucleotide hybridization and ligated into plasmid
vectors pTFMa or pF119 according to published procedures (24). The
recombinant plasmids were linearized by restriction enzymes
NsiI (pTFMa) or FokI (pF119) and transcribed
in vitro by T7 RNA polymerase. Point mutations in the gene
were introduced by site-directed mutagenesis using the Mutagene
(Bio-Rad) or QuickChange (Strategene) kits and the gene sequences were
confirmed by dideoxy sequencing. T7 transcripts of the wild-type and
mutant genes were purified by denaturing 12%, 7 M urea
polyacrylamide gel electrophoresis.
Preparation of Recombinant M. jannaschii
ProRS--
The gene for M. jannaschii ProRS, identified
from the genomic data base (25), was isolated by polymerase chain
reaction amplification of the genomic DNA. The amplified gene
was cloned into the pET-19 vector (Novagen) behind the sequence
encoding the histidine tag, and errors that arose from the polymerase
chain reaction were corrected by site-directed mutagenesis. The
sequence of the final cloned gene was confirmed by dideoxy sequencing
of the entire open reading frame. Expression of the recombinant gene was achieved in E. coli BL21(DE3) grown to an
A600 of 0.4-0.6 and induced with
0.1-0.3 mM
isopropyl-1-thio- Aminoacylation with Proline--
Proline and
3H-labeled proline (100 Ci/mmol) were purchased from Sigma
and PerkinElmer Life Sciences, respectively. Assays were carried out at
60 or 65 °C under conditions described previously (23).
Aminoacylation activity of tRNA mutants with substitutions in the
acceptor end was measured with 2.5 nM ProRS and 1.0-16.0 µM tRNA. Values reported are the average of at least
three determinations with an average standard deviation of 23.8%.
Aminoacylation activity of tRNA mutants with substitutions in the
anticodon was measured using 5 nM ProRS and 0.5 µM of a tRNA transcript, which was below the estimated
Km of the tRNA. Preliminary studies indicated that
the Km for the wild-type transcript was 1.0 µM (under the experimental conditions) and that of
mutants containing substitutions in the anticodon was in the range of
3-6 µM.2
Values reported are the average of at least two determinations, which
differed by less than 6.1%.
Sequence Analysis--
Multiple sequence alignments were
performed using the PILEUP program provided by the Genetics Computer
Group (Madison, WI). The value of 2.600 was used for the gap creation
penalty. Sequences used for alignments were obtained from ProRS
sequences in public data bases.
M. jannaschii ProRS Is a Eukaryotic-like
Enzyme--
Multiple sequence alignments of ProRS, including members
from all three domains of life, have divided the enzymes into two distinct groups (13, 14). M. jannaschii ProRS is a member of
the eukaryotic-like group (Fig. 1), which
also includes all eukaryotic enzymes, some eubacterial enzymes (such as
mycoplasma and spirochetes), and T. thermophilus ProRS. The
x-ray crystal structure of the T. thermophilus enzyme is
known and is shown in Fig. 1. The prokaryotic-like group of ProRS
includes the majority of the eubacterial enzymes as well as the
mitochondrial enzymes of eucarya. Although there is no known
three-dimensional structure of a ProRS from this group, an analysis of
the alignments, in addition to the crystal structure of T. thermophilus ProRS, reveal clear structural differences between
the two groups. Enzymes of the eukaryotic-like group have an extra
C-terminal domain of about 80 residues beyond the anticodon-binding
domain. In the structure of T. thermophilus ProRS, this
domain binds one atom of zinc, and its extreme C terminus folds back
into the conserved catalytic core (14). In contrast, the prokaryotic
enzymes have a large insertion domain (about 180 residues) of unknown
function between motif 2 and motif 3 (Fig. 1).
Recognition of tRNA by M. jannaschii ProRS--
The UGG
isoacceptor was used for studies of tRNA recognition by M. jannaschii ProRS (Fig. 2). This
isoacceptor contains three G:C base pairs at the extreme acceptor end
and an A73 discriminator base, all of which are conserved in the GGG
isoacceptor for tRNAPro (not shown). The features of the
acceptor stem in the two known isoacceptors of M. jannaschii
tRNAPro thus are notable for the presence of both C72 and
A73. This combination is distinct from that found in the corresponding
positions of E. coli and human tRNAPro (Fig.
4 below). Specifically, the acceptor stem
of all eubacterial proline tRNAs contains G72 and A73, both of which
are important for aminoacylation (6). In contrast, the acceptor stem of
all eukaryotic proline tRNAs contains C72 and C73, which play only a
minor role in aminoacylation (13). However, both the E. coli and human isoacceptors place a strong emphasis on recognition of the
anticodon nucleotides G35 and G36.
To compare the distribution of M. jannaschii
tRNAPro specificity determinants with those of E. coli and human, mutations were introduced into the acceptor stem
and the anticodon. Variants containing a single substitution of C72 or
A73, or of one of the anticodon nucleotides, were created by
site-directed mutagenesis and transcribed in vitro by T7 RNA
polymerase. Each variant was heat-denatured and refolded into its
native state in the presence of Mg2+. Steady-state
aminoacylation assays were then carried out, and the catalytic
efficiency (expressed as
kcat/Km) of each mutant
relative to that of the wild-type tRNA was determined.
As shown in Fig. 2, substitution of A73 in M. jannaschii
tRNAPro by any of the other three nucleotides has a small
effect (about 10-fold) on
kcat/Km. The G73 substitution
has the most severe effect (12.2-fold), whereas the C73 or U73
substitution has a smaller effect (2-fold and 6-fold, respectively).
The relative decreases in activity are also shown in Fig.
3A, where the initial rates of aminoacylation of the C73,
U73, and G73 mutants relative to that of the wild-type are compared.
The effects at A73 are minor compared with those previously observed in
the E. coli system. In E. coli
tRNAPro, substitution of A73 with G had a greater than
100-fold effect on aminoacylation, and substitution with the two
pyrimidine nucleotides U73 and C73 resulted in 30-40-fold decreases in
kcat/Km, respectively (6).
Thus, A73 is much less significant for M. jannaschii tRNA
recognition than it is for the E. coli tRNA. The weak
contribution of A73 to recognition by M. jannaschii ProRS, however, is comparable with that of C73 in the human system.
Substitution of C73 in the human tRNA with A or G only had an ~2-fold
effect (13).
Position 72 of M. jannaschii tRNAPro also has a
minor role in aminoacylation (Fig. 2). Substitution with any other
nucleotide has a less than 10-fold effect on
kcat/Km. The U substitution results in the largest decrease (5.3-fold), followed by the G substitution (2.2-fold). The A substitution is well tolerated with only
a 1.4-fold decrease in
kcat/Km. Because of the small
effects observed at position 72, additional mutations involving
position 1 that would maintain a 1:72 base pair in the acceptor stem
were not investigated. In contrast, position 72 is an important
specificity determinant for E. coli tRNAPro (6).
Substitution of G72 in E. coli tRNAPro with
either A or U reduces kcat/Km
by more than 150-fold, whereas substitution with C72 has an ~30-fold
effect. Similar to the M. jannaschii system, substitution of
C72 in human tRNAPro does not negatively impact
aminoacylation by the human enzyme (13).
We next tested mutations in all three anticodon positions of M. jannaschii tRNAPro for their effect on aminoacylation
catalytic efficiency. As expected, substitution of U34 with A or G had
no effect on aminoacylation, whereas substitution with C resulted in
only a 1.3-fold decrease (Fig. 2). The absence of an effect at position
34 is in accordance with results previously obtained in the E. coli and human tRNAs.
The anticodon nucleotide G35 of M. jannaschii
tRNAPro is a minor determinant for aminoacylation.
Substitution of G35 with A has the largest effect (9-fold), followed by
the C substitution (8-fold), and the U substitution (1.5-fold) (Fig.
2). The magnitude of these effects is similar to those observed in the
E. coli tRNA but is minor compared with those in the human
tRNA. In the latter case, the substitution of G35 with any other
nucleotides results in a large decrease in catalytic efficiency
(120-460-fold) (13).
Of all the positions tested in this study, the most important
specificity determinant in M. jannaschii tRNAPro
is G36. Substitution with C alone reduces the
kcat/Km of aminoacylation by
250-fold, and substitution with A results in a nearly 40-fold decrease
(Fig. 2). The exception is the U substitution, which maintains a
kcat/Km that is similar to
that of the wild-type tRNA. A representative aminoacylation assay of
the C36, A36, and U36 mutants relative to that of the wild-type
(initial rate data) is also shown in Fig. 3B. Interestingly, although the magnitude of the effects observed upon mutagenesis of G36
differs somewhat among the three tRNA species (E. coli, human, and M. jannaschii), the order of sensitivity to the
different nucleotide substitutions is the same. That is, in all cases,
substitution with C has the most severe effect, followed by A, and then U.
The relative importance of the acceptor stem and anticodon recognition
elements in E. coli, human, and M. jannaschii
tRNAs is summarized in Fig. 4. Although
the nucleotides at position 72 and 73 in the acceptor stem are major
determinants in E. coli tRNA, they are only minor
determinants in M. jannaschii tRNA and are even less
important in human tRNA. Conversely, although G35 in the anticodon is
the most important determinant in the human tRNA, it is a minor
determinant in both the E. coli and M. jannaschii systems. However, all three tRNAs maintain a strong emphasis on G36 as
a specificity determinant, and in the case of M. jannaschii tRNA, it is the only nucleotide tested that makes a major contribution to aminoacylation. Thus, as with the human system, anticodon
recognition is more important than acceptor stem recognition for
M. jannaschii ProRS. However, the details of anticodon
recognition appear to be more similar to that of the E. coli
system. For both E. coli and M. jannaschii ProRS
enzymes, position 36 is the primary determinant, whereas for the human
enzyme, position 35 is more critical (Fig. 4). From these data, we
conclude that there is a shift of emphasis in synthetase recognition
from the prokaryotic to the eukaryotic systems. In the former, both
acceptor stem and anticodon recognition play approximately equal roles,
whereas in the latter, anticodon recognition dominates. Also, we
conclude that, within the eukaryotic-like group, there are differences
in the details of anticodon recognition. Whereas G35 is the major
determinant in the anticodon of the human tRNA, G36 recognition is more
important in M. jannaschii tRNA, a trend that is shared by
the prokaryotic E. coli tRNA (Fig. 4).
Co-adaptation of Synthetase Motifs with Changes of tRNA Specificity
Determinants--
The shift in location of tRNA specificity
determinants from the prokaryotic (acceptor stem and anticodon) to the
eukaryotic systems (primarily anticodon) prompted a closer examination
of sequences in the motif 2 loop of the respective synthetases.
Specific sequences within the motif 2 loop of many class II
synthetases, including E. coli ProRS, have been identified
as important for recognition of the acceptor stem. In particular, the
RPR sequence, conserved in all eubacterial ProRS enzymes, is critical
for aminoacylation by E. coli ProRS (12). Substitution of
the first R in this sequence (R144) reduces
kcat/Km of aminoacylation by
more than 1000-fold but does not affect amino acid activation,
indicating a role exclusively in tRNA recognition. Cross-linking
experiments also confirmed that this R is proximal to the critical G72
nucleotide of the E. coli tRNAPro acceptor stem.
In contrast, mutation of the corresponding position in the human enzyme
(K1084) had no effect on human tRNAPro aminoacylation, a
result that is in accordance with the lack of base-specific acceptor
stem recognition in the eukaryotic system (12).
An extensive sequence alignment of the motif 2 loop of ProRS from 54 species, consisting of diverse members of the prokaryotic-like and
eukaryotic-like groups, was performed. A representative subset of the
aligned sequences is presented in Fig. 5.
Although the RPRXG motif is strictly conserved within the
prokaryotic-like enzymes, it is absent from the eukaryotic-like group
and is replaced by the less conserved KHXXP motif. For
example, M. jannaschii ProRS has the sequence KHTRP, whereas
the corresponding sequence in the human enzyme is KHPQP. Although the
function of the KHXXP motif is not entirely clear,
mutagenesis of individual amino acids within this sequence of the human
enzyme had little effect on aminoacylation (12). Based on the
relatively minor role of tRNAPro acceptor stem recognition
by M. jannaschii ProRS (Fig. 1), a similar result is
predicted for this system, and experiments to test this hypothesis are
under way. Thus, the shift of tRNA specificity determinants from the
acceptor end in E. coli tRNAPro to the anticodon
end in the human tRNA is accompanied by changes in protein motifs that
recognize the acceptor end. The M. jannaschii enzyme is
segregated with the human enzyme in the sequence alignment, and this
segregation is consistent with the weak of tRNA acceptor end
recognition.
In contrast to acceptor stem recognition, the pattern of anticodon
recognition by M. jannaschii ProRS is more similar to that of the E. coli enzyme than that of the human enzyme. Both
the M. jannaschii and E. coli enzymes emphasize
G36 more than G35, whereas the opposite holds true for the human
enzyme. However, despite functional similarity with the E. coli enzyme, sequence motifs in the anticodon-binding domain of
the M. jannaschii enzyme more closely resemble those of the
human synthetase (Fig. 6). For example,
the M. jannaschii and human enzymes share the conserved RXE motif, where R is position 336 and 1294 in M. jannaschii and human ProRS, respectively. This residue is believed
to contact G36 in the tRNA anticodon, as predicted by the co-crystal
structure of T. thermophilus ProRS complexed with the
anticodon domain of tRNAPro (15). The E. coli
enzyme instead has the sequence TIV (where T is at position 531). Also,
although the M. jannaschii and human enzymes share the
conserved EXXX(R/K)D motif (E338 in M. jannaschii ProRS and E1296 in human ProRS), which is believed to contact G35 in
the anticodon, the E. coli enzyme has the VXXXRN
motif (V533 in E. coli ProRS). Thus, although the M. jannaschii enzyme has remained segregated with the human enzyme in
its anticodon-binding sequence, the details of anticodon recognition
appear to have diverged.
The functional divergence in anticodon recognition by M. jannaschii ProRS, despite the sequence similarity with the
eukaryotic-like group, may be because of the requirement of the
archaeal enzyme to also recognize and specifically aminoacylate
tRNACys with cysteine. The anticodon of M. jannaschii tRNACys is GCA, which is significantly
different from the (U/G)GG anticodon of tRNAPro. The lack
of strong recognition at position 35, which is opposite from that of
the human enzyme (Fig. 4), may be important for its unusual dual
recognition capability. The molecular basis for how M. jannaschii ProRS recognizes tRNACys is currently being investigated.
In summary, the investigation of tRNA recognition by the archaeal
M. jannaschii ProRS and comparison of this recognition with that of the E. coli and human enzymes have provided evidence
of divergent adaptation between the tRNA and synthetase. The archaeal enzyme parallels the human enzyme with regard to acceptor stem recognition, in agreement with their sequence similarity in the motif 2 loop region. In particular, the minor role of A73 in recognition by
M. jannaschii ProRS is similar to the lack of recognition of C73 by the human enzyme. These results are in contrast to the strong
recognition of this discriminator base nucleotide (A73), as well as
G72, by the E. coli enzyme. With regard to recognition of
the anticodon, the archaeal enzyme has diverged from the human enzyme,
despite their sequence similarity. One manifestation of the divergence
is the shift of focus from G35 to G36. The diminished role of G35 for
the archaeal enzyme may reflect its dual function in recognition of
both tRNAPro and tRNACys, which contains C35.
The mechanistic details of this dual specificity remain to be elucidated.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-strands of motif 2 (12). Specific amino acid residues within the
motif 2 loop are known to make critical acceptor stem contacts in many
class II synthetases including E. coli ProRS (12, 16-20).
The prokaryotic-like group of ProRS enzymes differs from the
eukaryotic-like group by another major feature. Whereas the prokaryotic
enzymes contain an insertion sequence between motif 2 and motif 3, the
eukaryotic group lacks this insertion and instead contains a longer
C-terminal extension (Fig. 1) (13).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-galactopyranoside for 3-4 h at
37 °C. The recombinant ProRS was soluble and well expressed and was
purified using the Co2+-chelated Talon resin
(CLONTECH) or Ni2+-NTA resin (Qiagen)
according to the manufacturer's instructions. The concentration of the
purified ProRS was determined based on active-site titration using the
adenylate burst assay (26).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (30K):
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Fig. 1.
Top, ribbon diagram of the
crystal structure of T. thermophilus ProRS. Only the monomer
is shown, although the protein exists as a homodimer. The enzyme is a
class IIa aminoacyl-tRNA synthetase, and its conserved domains
containing motifs 1-3 are yellow. The insertion between
motif 2 and motif 3 is green, and the portion unique to the
prokaryotic-like group is dark blue. The anticodon-binding
domain is blue, and the extended C-terminal domain that
folds back to the active site is purple. Bottom,
schematic diagram of the architecture of the two groups of ProRS based
on sequence alignment of 54 sequences. The color notations are the same
as those shown in the crystal structure of T. thermophilus
ProRS. The eukaryotic-like group is represented by M. jannaschii, human, and T. thermophilus enzymes, and the
prokaryotic-like group is represented by E. coli and
mitochondrial D. melanogaster enzymes. The human enzyme in
the eukaryotic-like group has an extended N-terminal domain
(light blue).
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Fig. 2.
Sequence and cloverleaf structure of the UGG
isoacceptor of M. jannaschii tRNAPro
(unmodified) and variant transcripts prepared and tested in this
study. Nucleotide changes that were tested are indicated by
arrows, and the numbers in parentheses are the
change (x-fold) in
kcat/Km relative to that of
the wild-type transcript.
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Fig. 3.
A, comparison of a representative set of
initial rates of aminoacylation by M. jannaschii ProRS (5 nM) with the wild-type (wt), A73C, A73U, and
A73G variants of M. jannaschii tRNAPro
transcript (0.2 µM each). B, comparison of a
representative set of initial rates of aminoacylation by M. jannaschii ProRS (5 nM) with the wild-type, G36U,
G36A, and G36C variants of the tRNA transcript (0.5 µM
each). Data were fitted to an exponential curve or line.
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Fig. 4.
The sequence and cloverleaf structures of
E. coli, human, and M. jannaschii
tRNAPro. Acceptor stem (boxed) and
anticodon nucleotide (circled) positions tested in this and
previous work are indicated. The arrows point to nucleotides
that are the specificity determinants for the respective cognate
synthetase, as determined by in vitro aminoacylation
kinetics. The absence of an arrow indicates that the position is not a
recognition element. The size of an arrow indicates the relative
importance of the element as follows. A small arrow
indicates a minor (2-12-fold) effect, a medium arrow
indicates that at least one mutation tested had a large (>100-fold)
effect, and a large arrow indicates that all three mutations
had a >100-fold effect on the
kcat/Km of
aminoacylation.
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Fig. 5.
Alignment of the motif 2 region of
representative ProRS sequences, including 10 sequences of the
prokaryotic-like group (blue), 9 sequences of the
archaeal division of the eukaryotic-like group
(brown), and 12 sequences of the eukaryotic division
of the eukaryote-like group (green). Residues of
complete identity between the two groups are shaded.
Residues within the motif 2 loop that are conserved among the
prokaryotic-like group (the RPRXG motif) or semi-conserved
among the eukaryotic-like group (the KHXXP motif) are
shaded and are also indicated by a red box. The
arrows above the alignment indicate the -sheet
structure.
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Fig. 6.
Alignment of the anticodon-binding region of
representative ProRS sequences, where the color scheme is similar to
that in Fig. 5. Residues of identity between the two groups are
shaded, whereas residues predicted to be involved in binding
G35 and G36 within each group are shaded and
boxed (14, 15). Arrows and cylinders
indicate -sheets and
-helices, respectively.
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ACKNOWLEDGEMENTS |
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We thank Hiromi Motegi for technical support.
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FOOTNOTES |
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* This work was funded by National Institutes of Health Grants GM49928 (to K. M.-F.) and GM56662 (to Y.-M. H.) and by a grant from the Ministry of Education, Science, and Culture, Japan (to K. S.). In addition, partial support of the research was provided by the National Science Foundation (MCB-9904956 to Y.-M. 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.
These authors contribute equally to this work.
¶ Supported by a National Institutes of Health Molecular Biophysics Training Grant.
Supported by the American Heart Association,
Pennsylvania-Delaware Affiliate.
§§ To whom correspondence may be addressed. Tel.: 612-624-0286; Fax: 612-626-7541; E-mail: musier@chem.umn.edu.
¶¶ To whom correspondence may be addressed. Tel.: 215-503-4480; Fax: 215-923-9162; E-mail: Ya-Ming.Hou@mail.tju.edu.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M100465200
2 J. Wang and Y.-M. Hou, unpublished observation.
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ABBREVIATIONS |
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The abbreviation used is: ProRS, prolyl-tRNA synthetase.
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REFERENCES |
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