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PubMed2003

tRNA requirements for glyQS antitermination: a new twist on tRNA.

Transcription antitermination of the Bacillus subtilis glyQS gene, a member of the T box gene regulation family, can be induced during in vitro transcription in a minimal system using purified B. subtilis RNA polymerase by the addition of unmodified T7 RNA polymerase-transcribed tRNA(Gly). Antitermination was previously shown to depend on base-pairing between the glyQS leader and the tRNA at the anticodon and acceptor ends. In this study, variants of tRNA(Gly) were generated to identify additional tRNA elements required for antitermination activity, and to determine the effect of structural changes in the tRNA. We find that additions to the 3' end of the tRNA blocked antitermination, in agreement with the prediction that uncharged tRNA is the effector in vivo, whereas insertion of 1 nucleotide between the acceptor stem and the 3' UCCA residues had no effect. Disruption of the D-loop/T-loop tertiary interaction inhibited antitermination function, as was previously demonstrated for tRNA(Tyr)-directed antitermination of the B. subtilis tyrS gene in vivo. Insertion of a single base pair in the anticodon stem was tolerated, whereas further insertions abolished antitermination. However, we find that major alterations in the length of the acceptor stem are tolerated, and the insertions exhibited a pattern of periodicity suggesting that there is face-of-the-helix dependence in the positioning of the unpaired UCCA residues at the 3' end of the tRNA for interaction with the antiterminator bulge and antitermination.

Anticodon↗
PubMed1988

Biosynthesis of tRNA in yeast mitochondria. An endonuclease is responsible for the 3'-processing of tRNA precursors.

To study the mechanism involved in the 3'-processing of mitochondrial tRNA precursors, we examined tRNA processing in a reconstituted system with a yeast mitochondrial extract. Two mitochondrial tRNA(Glu) precursors synthesized from SP6 RNA polymerase-directed transcription system were used as substrates. One contained a 214-nucleotide 5' terminus and 115-123-nucleotide 3' trailer. The other had the same sized 3' trailer, but contained a mature 5' terminus. An endonucleolytic activity was identified in a mitochondrial S30 fraction which cleaves the 3' terminus of the latter tRNA precursor precisely at the in vivo CCA addition site. No cleavage of the 5'-extended precursor was observed in vitro. This mitochondrial 3'-processing activity was partially purified using DEAE-CL-6B chromatography. It removes the 3' trailer sequence from the 5'-matured precursor leaving a 3'-hydroxyl group on the processed tRNA and a 5'-phosphate group on the trailer. The resulting tRNA product serves as a substrate for tRNA nucleotidyltransferase which catalyzes the addition of CCA residues to the tRNA to complete its 3' maturation. Thus, yeast mitochondrial 3'-tRNA processing events resemble those found in eucaryotic cytoplasmic/nuclear systems where a single endonucleolytic cleavage is responsible for the formation of the 3' end of the tRNAs. This is in contrast to the multistep 3'-processing events known to occur in procaryotes.

Base Sequence↗
PubMed1986

A single mutation in loop IV of Escherichia coli SuIII tRNA blocks processing at both 5'- and 3'-ends of the precursor tRNA.

We showed previously that a single mutation (T54----A54) within the highly conserved GT psi C sequence in the Escherichia coli SuIII tRNA gene results in an Su- phenotype, and we presented preliminary evidence that this mutation affected biosynthesis of the tRNA. We now show that the A54 mutation has no effect on transcription but prevents accumulation of mature mutant tRNA. The absence of mature mutant tRNA is due to blocks in processing of the precursor tRNA and is not due to instability of the mature tRNA. Characterization of the A54 precursors (approximately 130, approximately 90, and approximately 85 nucleotides long), which accumulate in E. coli minicells, indicates multiple blocks to processing of mutant transcripts. All three size classes of precursors have heterogeneous 3'-termini and contain extensions of three to seven nucleotides beyond the 3'-CCA sequence of mature tRNA indicating either a block or delay in 3'-exonucleolytic processing. The approximately 130- and approximately 90-nucleotide-long precursors also have 5'-terminal extensions suggesting a block in the 5'-processing reaction catalyzed by RNase P. Only the approximately 85-nucleotide-long precursor, which represents a small fraction of the total tRNA precursors, has the correct 5'-end of the mature tRNA.

Base Sequence↗
PubMed1996

The growth defect in Escherichia coli deficient in peptidyl-tRNA hydrolase is due to starvation for Lys-tRNA(Lys).

The existence of a conditional lethal temperature-sensitive mutant affecting peptidyl-tRNA hydrolase in Escherichia coli suggests that this enzyme is essential to cell survival. We report here the isolation of both chromosomal and multicopy suppressors of this mutant in pth, the gene encoding the hydrolase. In one case, the cloned gene responsible for suppression is shown to be lysV, one of three genes encoding the unique lysine acceptor tRNA; 10 other cloned tRNA genes are without effect. Overexpression of lysV leading to a 2- to 3-fold increase in tRNA(Lys) concentration overcomes the shortage of peptidyl-tRNA hydrolase activity in the cell at non-permissive temperature. Conversely, in pth, supN double mutants, where the tRNA(Lys) concentration is reduced due to the conversion of lysV to an ochre suppressor (supN), the thermosensitivity of the initial pth mutant becomes accentuated. Thus, cells carrying both mutations show practically no growth at 39 degrees C, a temperature at which the pth mutant grows almost normally. Growth of the double mutant is restored by the expression of lysV from a plasmid. These results indicate that the limitation of growth in mutants of E.coli deficient in Pth is due to the sequestration of tRNA(Lys) as peptidyl-tRNA. This is consistent with previous observations that this tRNA is particularly prone to premature dissociation from the ribosome.

Base Sequence↗
PubMed1996

Specific cleavage of target RNAs from HIV-1 with 5' half tRNA by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can be converted to an RNA cutter that recognizes four bases, with about a 65-nt 3'-truncated tRNA(Arg) or tRNA(Ala). The 3'-truncated tRNA recognizes the target RNA via four base pairings between the 5'terminal sequence and a sequence 1-nt upstream of the cleavage site, resulting in a pre-tRNA-like complex (Nashimoto M, 1995, Nucleic Acids Res 23:3642-3647). Here I developed a general method for more specific RNA cleavage using 3' tRNase. In the presence of a 36-nt 5' half tRNA(Arg) truncated after the anticodon, 3' tRNase cleaved the remaining 56-nt 3' half tRNA(Arg) with a 19-nt 3' trailer after the discriminator. This enzyme also cleaved its derivatives with a 5' extra sequence or nucleotide changes or deletions in the T stem-loop and extra loop regions, although the cleavage efficiency decreases as the degree of structural change increases. This suggests that any target RNA can be cleaved site-specifically by 3'tRNase in the presence of a 5' half tRNA modified to form a pre-tRNA-like complex with the target. Using this method, two partial HIV-1 RNA targets were cleaved site-specifically in vitro. These results also indicate that the sequence and structure of the T stem-loop domain are important, but not essential, for the recognition of pre-tRNAs by 3' tRNase.

Animals↗
PubMed1990

Closely spaced and divergent promoters for an aminoacyl-tRNA synthetase gene and a tRNA operon in Escherichia coli. Transcriptional and post-transcriptional regulation of gltX, valU and alaW.

The transcription of the gltX gene encoding the glutamyl-tRNA synthetase and of the adjacent valU and alaW tRNA operons of Escherichia coli K-12 has been studied. The alaW operon containing two tRNA(GGCAla) genes, is 800 base-pairs downstream from the gltX terminator and is transcribed from the same strand. The valU operon, containing three tRNA(UACVal) and one tRNA(UUULys) (the wild-type allele of supN) genes, is adjacent to gltX and is transcribed from the opposite strand. Its only promoter is upstream from the gltX promoters. The gltX gene transcript is monocistronic and its transcription initiates at three promoters, P1, P2 and P3. The transcripts from one or more of these promoters are processed by RNase E to generate two major species of gltX mRNA, which are stable and whose relative abundance varies with growth conditions. The stability of gltX mRNA decreases in an RNase E- strain and its level increases with growth rate about three times more than that of the glutamyl-tRNA synthetase. The 5' region of these mRNAs can adopt a stable secondary structure (close to the ribosome binding site) that is similar to the anticodon and part of the dihydroU stems and loops of tRNA(Glu), and which might be involved in translational regulation of GluRS synthesis. The gltX and valU promoters share the same AT-rich and bent upstream region, whose position coincides with the position of the upstream activating sequences of tRNA and rRNA promoters to which they are similar. This suggests that gltX and valU share transcriptional regulatory mechanisms.

Amino Acyl-tRNA Synthetases↗
PubMed1984

The beta subunit of E. coli glycyl-tRNA synthetase plays a major role in tRNA recognition.

The contributions made by the alpha and beta subunits of E. coli glycyl-tRNA synthetase to the recognition of tRNA have been investigated via binding and immunological methods. Using the nitrocellulose filter assay, we have shown that isolated beta subunit, but not the alpha subunit, binds [14C]glycyl-tRNA with an affinity comparable to that of the native enzyme. Further, the data indicate that the beta subunit possesses one binding site for glycyl-tRNA while the native or reconstituted enzyme (alpha 2 beta 2) has two sites. Rabbit antibodies directed at the beta subunit or the holoenzyme inhibit efficiently the ability of the enzyme to aminoacylate tRNA while alpha-subunit antibodies have a smaller effect. Since none of the antisera have an appreciable effect on the ATP-PPi exchange activity of the enzyme under these conditions, the beta-subunit (and holoenzyme) antisera evidently interfere with productive tRNA binding. Taken together, the data indicate that the larger, beta subunit of glycyl-tRNA synthetase plays a major role in tRNA recognition.

Amino Acyl-tRNA Synthetases↗
PubMed1987

Expression of the aminoacyl-tRNA synthetase complex in cultured Chinese hamster ovary cells. Specific depression of the methionyl-tRNA synthetase component upon methionine restriction.

Cultured Chinese hamster ovary cells were subjected to amino acid restriction to examine its effects on the level of expression of the nine aminoacyl-tRNA synthetase components of the multienzyme complex which was previously characterized (Mirande, M., Le Corre, D., and Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289). Lowering the methionine concentration in the medium from 100 to 1 microM led to growth arrest, rapid deacylation of tRNAMet, and progressive 2-fold elevation of the methionyl-tRNA synthetase level, as assessed by specific activity measurements and immunotitration. The levels of the other eight aminoacyl-tRNA synthetases were not affected. Total methionine deprivation led to the additional derepression of the leucyl- and isoleucyl-tRNA synthetase components, whereas the corresponding tRNAs remained fully acylated. These pleiotropic responses to total methionine restriction were abolished in the presence of 2 mM methioninol, suggesting that amino acid transport systems may play a role in the regulation of aminoacyl-tRNA synthetase expression. The effect of total deprivation of arginine, glutamine, isoleucine, leucine, lysine, or proline from the culture medium on the level of expression of the corresponding aminoacyl-tRNA synthetases was also examined. In all cases, no elevation of the level of the corresponding synthetase was observed. The behavior of methionyl-tRNA synthetase from Chinese hamster ovary cells displaying a 2-fold increased level of the enzyme due to methionine restriction was examined in detail. Failure to detect a free form of the enzyme by gel filtration, as well as the finding that the isolated complex displayed twice the amount of methionyl-tRNA synthetase relative to the other components, indicates that this multienzyme structure can accommodate at least one additional copy of one of its components.

Acylation↗
PubMed1994

A motif in human histidyl-tRNA synthetase which is shared among several aminoacyl-tRNA synthetases is a coiled-coil that is essential for enzymatic activity and contains the major autoantigenic epitope.

In myositis, disease-specific autoantibodies may be directed against an aminoacyl-tRNA synthetase, usually histidyl-tRNA synthetase. To explore the basis for this phenomenon, we have made recombinant histidyl-tRNA synthetase in the baculovirus system. It was enzymatically active and recognized by human autoantibodies. A truncated protein lacking the first 60 amino acids was inactive as an antigen and as an enzyme. This region is within the first two exons, is predicted to have a coiled-coil configuration, and is found in some other synthetases but not in Escherichia coli or yeast histidyl-tRNA synthetase. Circular dichroism showed that the peptides from this region (amino acids 1-60 and 1-47) have the predicted high alpha-helical content, but smaller fragments (1-30, 14-45, and 31-60) do not. The peptides with a high alpha-helical content could inhibit autoantibodies almost completely, whereas the smaller peptides were unable to do so. The amino acid sequence of this coiled-coil domain in human histidyl-tRNA synthetase resembles the sequence of the extended this coiled-coil arm near the NH2 terminus of bacterial seryl-tRNA synthetase as well as similar regions in some eukaryotic aminoacyl-tRNA synthetases, raising the possibility that this domain serves a similar tRNA-stabilizing role and has been preserved from a common ancestor.

Amino Acid Sequence↗
PubMed1993

Overproduction and purification of native and queuine-lacking Escherichia coli tRNA(Asp). Role of the wobble base in tRNA(Asp) acylation.

Escherichia coli tRNA(Asp) was overproduced in E. coli up to 15-fold from a synthetic tRNA(Asp) gene placed in a plasmid under the dependence of an isopropyl-beta,D-thiogalactopyranoside-inducible promoter. Purification to nearly homogeneity (95%) was achieved after two HPLC DEAE-cellulose columns. E. coli tRNA(Asp)[G34] (having guanine instead of queuine at position 34) was obtained by the same procedure except that it was overproduced in a strain lacking the enzyme responsible for queuine modification. Nucleoside analysis showed that, except for the replacement of Q34 by G34 in mutant-derived tRNA(Asp), the base modification levels of both tRNAs are the same as those in wild-type E. coli tRNA(Asp). Kinetic properties of tRNA(Asp)[Q34] and [G34] with yeast AspRS compared to those in the homologous reactions in yeast and E. coli clearly indicate that the major identity elements are the same in both organisms: the conserved discriminant base and the anticodon triplet. In connection with this, we explored by site-directed mutagenesis the functional role of the interactions which, as revealed by the crystallographic structure, occur between the wobble base of yeast tRNA(Asp) and two residues of yeast AspRS. Their absence strongly affected aspartylation and the kd of tRNA(Asp). Each contact individually restores almost completely the wild-type acylation properties of the enzyme; thus, wobble base recognition in yeast appears to be more protected against mutational events than in E. coli, where only one contact is thought to occur at position 34.

Anticodon↗
PubMed1995

Evolution of tRNA recognition systems and tRNA gene sequences.

The aminoacylation of tRNAs by the aminoacyl-tRNA synthetases recapitulates the genetic code by dictating the association between amino acids and tRNA anticodons. The sequences of tRNAs were analyzed to investigate the nature of primordial recognition systems and to make inferences about the evolution of tRNA gene sequences and the evolution of the genetic code. Evidence is presented that primordial synthetases recognized acceptor stem nucleotides prior to the establishment of the three major phylogenetic lineages. However, acceptor stem sequences probably did not achieve a level of sequence diversity sufficient to faithfully specify the anticodon assignments of all 20 amino acids. This putative bottleneck in the evolution of the genetic code may have been alleviated by the advent of anticodon recognition. A phylogenetic analysis of tRNA gene sequences from the deep Archaea revealed groups that are united by sequence motifs which are located within a region of the tRNA that is involved in determining its tertiary structure. An association between the third anticodon nucleotide (N36) and these sequence motifs suggests that a tRNA-like structure existed close to the time that amino acid-anticodon assignments were being established. The sequence analysis also revealed that tRNA genes may evolve by anticodon mutations that recruit tRNAs from one isoaccepting group to another. Thus tRNA gene evolution may not always be monophyletic with respect to each isoaccepting group.

Amino Acyl-tRNA Synthetases↗
PubMed1991

Cysteinyl-tRNA synthetase is a direct descendant of the first aminoacyl-tRNA synthetase.

The gene encoding the cysteinyl-tRNA synthetase of E. coli was cloned from an E. coli genomic library made in lambda 2761, a lambda vector which can integrate and which carries a chloramphenicol resistance gene. A thermosensitive cysS mutant of E. coli was lysogenised and chloramphenicol-resistant colonies able to grow at 42 degrees C were selected to isolate phages containing the wild-type cysS gene. The sequence of the gene was determined. It codes for a 461 amino-acid protein and includes the sequences HIGH and KMSK known to be involved in the ATP and tRNA binding respectively of class I synthetases. The cysteinyl enzyme has segments in common with the cytoplasmic leucyl-tRNA synthetase of Neurospora crassa, the tryptophanyl-tRNA synthetase of Bacillus stearothermophilus, and the phenylalanyl-tRNA synthetase of Saccharomyces cerevisiae. Sequence comparisons show that the amino end of the cysteinyl-tRNA synthetase has similarities with prokaryotic elongation factors Tu; this region is close to the equivalent acceptor binding domain of the glutaminyl-tRNA synthetase of E. coli. There is a further similarity with the seryl enzyme (a class II enzyme) which has led us to propose that both classes had a common origin and that this was the ancestor of the cysteinyl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1990

Yeast methionyl-tRNA synthetase: analysis of the N-terminal extension and the putative tRNA anticodon binding region by site-directed mutagenesis.

Yeast methionyl-tRNA synthetase has a long N-terminal extension fused to the mononucleotide binding fold that occurs at the N-terminal end of the homologous E coli enzyme. We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame. The results show that 185 amino acids are dispensable for activity and stability. Removal of the next 5 residues affects the activity of the enzyme. The effect is more pronounced on the tRNA amino-acylation steps than on the adenylate formation step. The Km for ATP and methionine are unaltered, indicating that the global structure of the enzyme is maintained. The Km for tRNA increased slightly by a factor of 3, which indicates that the positioning of the tRNA on the surface of the molecule is not affected. There is, however, a great effect on the Vmax of the enzyme. Examination of the 3-D structure of the homologous E coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide binding fold does not participate directly in the catalytic cleft. It could, however, act at a distance by propagating a mutational alteration of the catalytic residues. The tRNA(Met) anticodon binding region of the E coli enzyme has recently been characterized. By mutagenesis of the topologically equivalent region in the yeast enzyme, we could identify residues that alter specifically the aminoacylation of the tRNA. Leu 658 provides a van der Waals contact that is critical for the recognition of the yeast tRNA.

Adenosine Triphosphate↗
PubMed1993

A 15N-1H nuclear magnetic resonance study on the interaction between isoleucine tRNA and isoleucyl-tRNA synthetase from Escherichia coli.

Imino 15N and 1H resonances of Escherichia coli tRNA(lIle) were observed in the absence and presence of E coli isoleucyl-tRNA synthetase. Upon complex formation of tRNA(lIle) with isoleucyl-tRNA synthetase, some imino 15N-1H resonances disappeared, and some others were significantly broadened and/or shifted in the 1H chemical shift, while the others were observed at the same 15N-1H chemical shifts. It was indicated that the binding of tRNA(lIle) with IleRS affect the following four regions: the anticodon stem, the junction of the acceptor and T stems, the middle of the D stem, and the region where the tertiary base pair connects the T, D, and extra loops. This result is consistent with those of chemical footprinting and site-directed mutagenesis studies. Taken together, these three independent results reveal the recognition mechanism of tRNA(lIle) by IleRS: IleRS recognizes all the identity determinants distributed throughout the tRNA(lIle) molecule, which induces changes in the secondary and tertiary structures of tRNA(lIle).

Base Sequence↗
PubMed2006

Active-site assembly in glutaminyl-tRNA synthetase by tRNA-mediated induced fit.

Structure-based mutational analysis was employed to probe an unusual intramolecular interaction between partially buried glutamate residues adjacent to the active site of Escherichia coli glutaminyl-tRNA synthetase (GlnRS). The crystal structures of unliganded GlnRS and the GlnRS-tRNA(Gln) complex reveal that the Glu34 and Glu73 side chain carboxylates contact each other only in the tRNA-bound state and that the interaction is formed via mutual induced-fit transitions that occur en route to the ground-state Michaelis complex. Steady-state and transient kinetic analysis of mutant enzymes suggest that the formation of this intermolecular contact is a key event that facilitates the proper formation of the active site. Mutants at both positions destabilize the binding of the substrate glutamine at the opposite side of the active-site cleft, whereas Glu73 appears to play an additional important role by promoting the correct binding of the 3'-acceptor end of tRNA adjacent to both ATP and glutamine. The data suggest the existence of multiple structural pathways by which the binding of tRNA propagates conformational transitions leading to the proper formation of the glutamine binding site. The single-turnover kinetic analysis also establishes that the Glu34 carboxylate does not play a direct enzymatic role as a catalytic base to help deprotonate the tRNA-A76 nucleophilic 2'-hydroxyl group. The elimination of this previously proposed mechanism, together with recent chemical modification experiments in the histidyl-tRNA synthetase system, emphasizes that substrate-assisted catalysis by the phosphate of the aminoacyl adenylate may be a common means by which all tRNA synthetases facilitate the aminoacyl transfer step of the reaction.

Amino Acyl-tRNA Synthetases↗
PubMed1996

Identity of prokaryotic and eukaryotic tRNA(Asp) for aminoacylation by aspartyl-tRNA synthetase from Thermus thermophilus.

The aspartate identity of tRNA for AspRS from Thermus thermophilus has been investigated by kinetic analysis of the aspartylation reaction of different tRNA molecules and their variants as well as of tRNAPhe variants with transplanted aspartate identity elements. It is shown that G10, G34, U35, C36, C38, and G73 determine recognition and aspartylation of yeast and T.thermophilus tRNA(Asp) by the thermophilic AspRS. This set of nucleotides specifies also tRNA aspartylation in the homologous yeast and Escherichia coli systems. Structural considerations indicate that the major aspartate identity elements interact with amino acids conserved in all AspRSs. It follows that the structural features of tRNA and synthetase specifying aspartylation are mainly conserved in various structural contexts and in organisms adapted to different life conditions. Mutations of tRNA identity elements provoke drastic losses of charging in the heterologous system involving yeast tRNA(Asp) and T. thermophilus AspRS. In the homologous systems, the mutational effects are less pronounced. However, effects in E. coli and T. thermophilus exceed those in yeast which are particularly moderate, indicating variations in the individual contributions of identity elements for aspartylation in prokaryotes and eukaryotes. Analysis of multiple tRNA mutants reveals cooperativity between the cluster of determinants of the anticodon loop and the additional determinants G10 and G73 for efficient aspartylation in the thermophilic system, suggesting that conformational changes trigger formation of the functional tRNA/synthetase complex.

Amino Acid Sequence↗
PubMed1997

Interaction of tRNA with tRNA (guanosine-1)methyltransferase: binding specificity determinants involve the dinucleotide G36pG37 and tertiary structure.

The sequence G37pG36 is present in all tRNA species recognized and methylated by the Escherichia coli modification enzyme tRNA (guanosine-1)methyltransferase. We have examined whether this dinucleotide sequence provides the base specific recognition signal for this enzyme and have assessed the role of the remaining tRNA in recognition. E. coli tRNAHis and yeast tRNAAsp were substituted with G at positions 36 and 37 and were found to be excellent substrates for methylation. This suggested that the general tRNA structure can be specifically bound by the enzyme. In addition, heterologous tRNA species including fully modified tRNA1Leu are excellent inhibitors of tRNA1Leu transcript methylation. Analyses of structural variants of yeast tRNAAsp and E. coli tRNA1Leu demonstrate clearly that the core tertiary structures of tRNA are required for recognition and that G37 must be in the correct position in space relative to important contacts elsewhere in the molecule. This latter conclusion was reached because the addition of one to three stacked base pairs in the anticodon stem of tRNA1Leu dramatically alters activity. In this case, the G37 base is rotated away from the correct position in space relative to other tRNA contact sites. The acceptor stem structure is required for optimal activity since deletion of three or five base pairs is detrimental to activity; however, specific base sequence may not be important because (i) the addition of three stacked base pairs of different sequence had little effect on activity and (ii) heterologous tRNAs with little or no sequence homology in the acceptor stem are excellent substrates. Both poly G and GpG are potent and specific inhibitors of enzyme activity and are minimal substrates which can be methylated, forming m1G. Taken together, these studies suggest that 1MGT can bind the general tRNA structure and that the crucial base-pair contacts are G37 and G36.

Anticodon↗
PubMed1999

Different adaptations of the same peptide motif for tRNA functional contacts by closely homologous tRNA synthetases.

The N73 nucleotide at the end of the tRNA acceptor stem is commonly used by tRNA synthetases for discrimination. Because only a few synthetase-tRNA cocrystal structures have been determined, understanding of the molecular basis for N73 discrimination is limited. Here we investigated the possibility that, for at least some synthetases, the capacity to recognize different N73 nucleotides resides in the variable sequence of the loop of motif 2, a motif found in all class II enzymes. In the cocrystal of the class II yeast aspartyl-tRNA synthetase, atomic groups of the G73 discriminator of tRNAAsp interact with three side chains of the enzyme. We examined lysyl-tRNA synthetase, a close structural homologue of the aspartyl enzyme. Different substitutions were introduced into the Escherichia coli enzyme (A73 discriminator) to make its loop more like that of the human enzyme (G73 discriminator). Our data show that the loop of motif 2 of the lysine enzyme makes tRNA functional contacts, as predicted from the structural comparison. And yet, the E. coli enzyme with the "humanized" loop sequence had the same quantitative kinetic preference for A73 versus G as the wild-type enzyme. We conclude that discriminator base selectivity in the lysine enzyme requires residues in addition to or other than those in the loop of motif 2. Thus, even tRNA synthetases that are close structural homologues may use the same RNA binding element to make functional contacts with places (in the acceptor stem) that are idiosyncratic to each synthetase-tRNA pair.

Acylation↗