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PubMed1990

Mapping of the active site of Escherichia coli methionyl-tRNA synthetase: identification of amino acid residues labeled by periodate-oxidized tRNA(fMet) molecules having modified lengths at the 3'-acceptor end.

Initiator tRNA molecules modified at the 3'-end and lacking either the A76 (tRNA-C75), the C75-A76 (tRNA-C74), the C74-C75-A76 (tRNA-A73), or the A73-C74-C75-A76 (tRNA-A72) nucleotides were prepared stepwise by repeated periodate, lysine, and alkaline phosphatase treatments. When incubated with trypsin-modified methionyl-tRNA synthetase (MTST), excess amounts of the dialdehyde derivative of each of these shortened tRNAs (tRNA-C75ox, tRNA-C74ox, tRNA-A73ox, and tRNA-A72ox) abolished both the isotopic [32P]PPi-ATP exchange and the tRNA aminoacylation activities of the enzyme. In the presence of limiting concentrations of the various tRNAox species, the relative extents of inactivation of the enzyme were consistent with the formation of 1:1 complexes of the reacting tRNAs with the monomeric modified synthetase. Specificity of the labeling was further established by demonstrating that tRNA-C75ox binds the enzyme with an equilibrium constant and stoichiometry values in good agreement with those for the binding of nonoxidized tRNA-C75. The peptides of MTST labeled with either tRNA-C75ox or tRNA-C74ox were identified. The chymotryptic digestion of the covalent MTST.[14C]tRNA-C75ox complex yielded four peptides (A-D). In the case of tRNA-C74ox, only two of the above peptides (C and D) were identified. Peptides A, B, C, and D corresponded to fragments Ser334-Phe340, Lys61-Leu65, Val141-Tyr165, and Glu433-Phe437, respectively, in the MTST primary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

A preferential role for lysyl-tRNA4 in the synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate by an arginyl-tRNA synthetase-lysyl-tRNA synthetase complex from rat liver.

The synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) can be catalyzed in vitro by a tetrameric tRNA synthetase complex from rat liver containing two lysyl-tRNA synthetase and two arginyl-tRNA synthetase subunits. This reaction required ATP, AMP, 50-100 microM zinc, and inorganic pyrophosphatase. We show here that AMP can be omitted from the reaction and that the zinc levels can be markedly reduced provided catalytic amounts of tRNA(Lys) are added to the reaction mixture. Ap4A synthesis with purified tRNA(Lys) isoacceptors showed that the minor species, tRNA(4Lys), was 3-fold more active than either of the two major tRNA(Lys) species, tRNA(2Lys) and tRNA(5Lys). No activity could be demonstrated with tRNA(Lys) from Escherichia coli or with tRNA(Lys) or tRNA(Phe) from yeast. Aminoacylation of tRNA(4Lys) was strictly required as determined by the fact that Ap4A synthesis was not observed until aminoacylation was nearly complete, inhibitors of aminoacylation blocked Ap4A synthesis, and there was a strict requirement for added lysine. None of the above observations could be demonstrated, however, when lysyl-tRNA(Lys) was directly supplied to the reaction mixture. Optimum Ap4A synthesis was obtained by the addition of 1 mol of tRNA(Lys)/mol of the synthetase complex. This reaction is unique because it does not require the prior formation of an aminoacyl-AMP intermediate and because it can actively synthesize Ap4A at physiological zinc concentrations. The preferential role for tRNA(4Lys) in Ap4A synthesis is consistent with its prior implication in cell division.

Adenine Nucleotides↗
PubMed1980

The plant aminoacyl-tRNA synthetases. 2'-DeoxyATP and ATP in reactions catalysed by yellow lupin aminoacyl-tRNA synthetases.

2'-Deoxyadenosine triphosphate (dATP) is not a substrate for valyl-tRNA, leucyl-tRNA and isoleucyl-tRNA synthetases from yellow lupin seeds. Yellow lupin seryl-tRNA, phenylalanyl-tRNA, tyrosyl-tRNA, arginyl-tRNA, lysyl-tRNA and methionyl-tRNA synthetases use dATP as a substrate both in aminoacyl deoxyadenylate formation and tRNA aminoacylation reactions. Generally, dATP is a poorer substrate, being 3 - 50% as effective as ATP. None of the other nucleoside triphosphates tested was shown to be a substrate for these enzymes. Specificity (k cat/Km) towards ATP is greater in tRNA aminoacylation than in pyrophosphate exchange reaction catalysed by seryl-tRNA synthetase. Energy of activation is the same (Ea = 18.5 kcal) for dATP- and ATP-dependent pyrophosphate exchange. Both dATP- and ATP-dependent tRNA aminoacylation reactions exhibit the same temperature dependence with Ea = 9.0 kcal. Half lives of the enzyme-bound seryl adenylate and deoxyadenylate are 2 min and 4 min, respectively (pH 8.0, 25 degrees C). Both enzyme-bound phenylalanyl adenylate and deoxyadenylate exhibit the same stability (half lives 0.3 min), and enzyme-bound tyrosyl deoxyadenylate is hydrolysed faster (half life 0.6 min) than tyrosyl adenylate (half life 2.5 min). Serine is transferred both from enzyme-bound seryl adenylate and deoxyadenylate to tRNA with the same 60% efficiency. One mol of ATP or 1.1 mol of dATP is hydrolysed per one mol of seryl-tRNA formed during aminoacylation in the complete ATP- or dATP-dependent system, respectively. AMP concentrations (up to 0.5 mM), which do not affect the equilibrium of the ATP-dependent tRNA esterification with serine, significantly change the equilibrium of the dATP-dependent reaction.

Adenosine Triphosphate↗
PubMed1992

Nucleotides of tRNA governing the specificity of Escherichia coli methionyl-tRNA(fMet) formyltransferase.

In Escherichia coli, the free amino group of the aminoacyl moiety of methionyl-tRNA(fMet) is specifically modified by a transformylation reaction. To identify the nucleotides governing the recognition of the tRNA substrate by the formylase, initiator tRNA(fMet) was changed into an elongator tRNA with the help of an in vivo selection method. All the mutations isolated were in the tRNA acceptor arm, at positions 72 and 73. The major role of the acceptor arm was further established by the demonstration of the full formylability of a chimaeric tRNA(Met) containing the acceptor stem of tRNA(fMet) and the remaining of the structure of tRNA(mMet). In addition, more than 30 variants of the genes encoding tRNA(mMet) or tRNA(fMet) have been constructed, the corresponding mutant tRNA products purified and the parameters of the formylation reaction measured. tRNA(mMet) became formylatable by the only change of the G1.C72 base-pair into C1-A72. It was possible to render tRNA(mMet) as good a substrate as tRNA(fMet) for the formylase by the introduction of a limited number of additional changes in the acceptor stem. In conclusion, A73, G2.C71, C3.G70 and G4.C69 are positive determinants for the specific processing of methionyl-tRNA(fMet) by the formylase while the occurrence of a G.C or C.G base-pair between positions 1 and 72 acts as a major negative determinant. This pattern appears to account fully for the specificity of the formylase and the lack of formylation of any aminoacylated tRNA, excepting the methionyl-tRNA(fMet).

Acyltransferases↗
PubMed2003

Probing the importance of tRNA anticodon: human immunodeficiency virus type 1 (HIV-1) RNA genome complementarity with an HIV-1 that selects tRNA(Glu) for replication.

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs at the primer binding site (PBS) that is complementary to the 3'-terminal nucleotides of tRNA(3)(Lys). Why all known strains of HIV-1 select tRNA(3)(Lys) for replication is unknown. Previous studies on the effect of altering the PBS of HIV-1 on replication identified an HIV-1 with a PBS complementary to tRNA(Glu). Since the virus was not initially designed to use tRNA(Glu), the virus had selected tRNA(Glu) from the intracellular pool of tRNA for use in replication. Further characterization of HIV-1 that uses tRNA(Glu) may provide new insights into the preference for tRNA(3)(Lys). HIV-1 constructed with the PBS complementary to tRNA(Glu) was more stable than HIV-1 with the PBS complementary to tRNA(Met) or tRNA(His); however, all of these viruses eventually reverted back to using tRNA(3)(Lys) following growth in SupT1 cells or peripheral blood mononuclear cells (PBMCs). New HIV-1 mutants with nucleotides in U5 complementary to the anticodon of tRNA(Glu) remained stable when grown in SupT1 cells or PBMCs, although the mutants grew more slowly than the wild-type virus. Sequence analysis of the U5 region and the PBS revealed additional mutations predicted to further promote tRNA-viral genome interaction. The results support the importance of the tRNA anticodon-genome interaction in the selection of the tRNA primer and highlight the fact that unique features of tRNA(3)(Lys) are exploited by HIV-1 for selection as the reverse transcription primer.

Anticodon↗
PubMed2003

[Seasonal differences in activity of tRNA and aminoacyl-tRNA synthetases of rabbit liver in myocardial ischemia].

UNLABELLED: The objective of this study was to examine the acceptor activities of tRNA for amino acids alanine and lysine and activities of corresponding aminoacyl-tRNA synthetases of normal rabbit liver 6, 12 and 24 h after experimental myocardial ischemia in different seasons of the year. MATERIAL AND METHODS: Male rabbits (2.5-3.5 kg) were used. Acute myocardial ischemia was induced by occlusion of the left anterior descending coronary artery. tRNA and aminoacyl-tRNA synthetases were isolated from normal (control) rabbit liver 6, 12 and 24 h after experimental myocardial ischemia in autumn (the months of September and October) and winter (the months of December and January). The acceptor activity of tRNA and activity of alanyl- and lysyl-tRNA synthetases were determined using 14C-labelled amino acids alanine and lysine. RESULTS: The results of study show that acceptor activity of rabbit liver tRNA for alanine and lysine under 6, 12 and 24 h experimental myocardial ischemia in autumn is higher by 18-52 percent than in winter. Activities of rabbit liver alanyl- and lysyl-tRNA synthetases under 6, 12 and 24 h experimental myocardial ischemia in autumn is less by 15-35 percent than in winter. No differences in activity of tRNA and aminoacyl-tRNA synthetases between normal groups of both seasons were observed. CONCLUSIONS: The experimental data suggest that there are differences in acceptor activity of rabbit liver tRNA for alanine and lysine and in activity of alanyl- and lysyl-tRNA synthetases after 6, 12 and 24 h experimental myocardial ischemia. Decreasing of acceptor activity of tRNA for alanine under experimental myocardial ischemia in winter correlate with increasing of activity of alanyl-tRNA synthetase. Decreasing of acceptor activity of tRNA for lysine under experimental myocardial ischemia correlate with increasing of lysyl-tRNA synthetase activity in both studied seasons. It may be part of the compensatory mechanism of the cell to keep synthesis of protein in normal range under extreme conditions.

Alanine↗
PubMed2003

Expanding tRNA recognition of a tRNA synthetase by a single amino acid change.

Aspartyl-tRNA synthetase (AspRS) occurs in two types: the discriminating enzyme (D-AspRS) forms only Asp-tRNA(Asp), whereas the nondiscriminating enzyme (ND-AspRS) also synthesizes Asp-tRNA(Asn), which is a required intermediate for protein synthesis in many organisms. We attempted to expand the tRNA recognition of the discriminating Thermococcus kodakaraensis AspRS to that of a ND-AspRS by in vitro mutagenesis. An alignment of 26 archaeal AspRS proteins revealed two positions (26 and 85 in the T. kodakaraensis sequence) whose amino acid identity changes according to the enzymes' tRNA specificity. In their anticodon-binding domain, D-AspRS proteins contain W26 (or Q26) and K85, compared with H26 and P85 in the ND-AspRSs. T. kodakaraensis AspRS gained the ability to form Asp-tRNA(Asn) in vitro when the W26H or K85P changes were introduced independently or in combination. In the aminoacylation of tRNA(Asn) or tRNA(Asp) transcripts, the mutant enzymes displayed at least a 100- to 500-fold change in tRNA specificity, as judged by the ratio of the k(cat)K(m) values of Asp-tRNA(Asp) vs. Asp-tRNA(Asn) formation. That T. kodakaraensis mutant AspRSs mischarge tRNA(Asn) was also manifested in the higher level (1.7%) of aspartylation of unfractionated Pyrococcus tRNA compared with that achieved by the wild-type enzyme (0.9%). Northern blot analysis of the Asp-tRNA separated by acidurea gel electrophoresis confirmed the in vitro synthesis of Asp-tRNA(Asn). A structure-based model points to a direct interaction of K85 in T. kodakaraensis AspRS with the anticodon nucleotide C36 of tRNA(Asp). Thus, a switch between D-AspRS and ND-AspRS enzymes could have evolved with only limited amino acid changes.

Amino Acid Sequence↗
PubMed2002

Functional dissection of the eukaryotic-specific tRNA-interacting factor of lysyl-tRNA synthetase.

In the cytoplasm of higher eukaryotic cells, aminoacyl-tRNA synthetases (aaRSs) have polypeptide chain extensions appended to conventional prokaryotic-like synthetase domains. The supplementary domains, referred to as tRNA-interacting factors (tIFs), provide the core synthetases with potent tRNA-binding capacities, a functional requirement related to the low concentration of free tRNA prevailing in the cytoplasm of eukaryotic cells. Lysyl-tRNA synthetase is a component of the multi-tRNA synthetase complex. It exhibits a lysine-rich N-terminal polypeptide extension that increases its catalytic efficiency. The functional characterization of this new type of tRNA-interacting factor has been conducted. Here we describe the systematic substitution of the 13 lysine or arginine residues located within the general RNA-binding domain of hamster LysRS made of 70 residues. Our data show that three lysine and one arginine residues are major building blocks of the tRNA-binding site. Their mutation into alanine led to a reduced affinity for tRNA(3)(Lys) or minimalized tRNA mimicking the acceptor-TPsiC stem-loop of tRNA(3)(Lys) and a decrease in catalytic efficiency similar to that observed after a complete deletion of the N-terminal domain. Moreover, covalent continuity between the tRNA-binding and core domain is a prerequisite for providing LysRS with a tRNA binding capacity. Thus, our results suggest that the ability of LysRS to promote tRNA(Lys) networking during translation or to convey tRNA(3)(Lys) into the human immunodeficiency virus type 1 viral particles rests on the addition in evolution of this tRNA-interacting factor.

Amino Acid Sequence↗
PubMed1990

Construction of Escherichia coli amber suppressor tRNA genes. III. Determination of tRNA specificity.

Using synthetic oligonucleotides, we have constructed a collection of Escherichia coli amber suppressor tRNA genes. In order to determine their specificities, these tRNAs were each used to suppress an amber (UAG) nonsense mutation in the E. coli dihydrofolate reductase gene fol. The mutant proteins were purified and subjected to N-terminal sequence analysis to determine which amino acid had been inserted by the suppressor tRNAs at the position of the amber codon. The suppressors can be classified into three groups on the basis of the protein sequence information. Class I suppressors, tRNA(CUAAla2), tRNA(CUAGly1), tRNA(CUAHisA), tRNA(CUALys) and tRNA(CUAProH), inserted the predicted amino acid. The class II suppressors, tRNA(CUAGluA), tRNA(CUAGly2) and tRNA(CUAIle1) were either partially or predominantly mischarged by the glutamine aminoacyl tRNA synthetase. The class III suppressors, tRNA(CUAArg), tRNA(CUAAspM), tRNA(CUAIle2), tRNA(CUAThr2), tRNA(CUAMet(m)) and tRNA(CUAVal) inserted predominantly lysine.

Amino Acid Sequence↗
PubMed1974

Cysteine activation in cultured cystinotic cells. The specific activity of cysteinyl-tRNA synthetase and tRNACys and the determination of the Michaelis-Menten constants for cysteinyl-tRNA synthetase.

This study explored the possibility whether an altered cysteinyl-tRNA synthetase might lead to the faulty regulation of cyst(e)ine levels in cystinotic cells. This hypotheses is attractive, since amino acid activation is important in the regulation of amino acid metabolism in microorganisms. By using cultured fibroblasts from patients with cystinosis, those cell components responsible for cysteine activation were examined: cyst(e)ine, the cysteinyl-tRNA levels, cysteinyl-tRNA synthetase activity, and the K(m) of cysteine, ATP, and tRNA(Cys) for cysteinyl-tRNA synthetase, Fibroblasts from two patients with the infantile form of cystinosis were labeled for three days with [(35)S]-cystine. In comparison with normal cells, these cells contained high levels of free cysteine and cystine. Labeled fibroblasts from a patient with the adolescent form of the disease contained elevated levels of cystine, although elevated cysteine levels were not detected. The ratio of acceptor activity of tRNA(Cys) to tRNA(Leu) in cystinotic cells was 0.46 in cystinotic cells and 0.54 in normal cells. The specific activity of cysteinyl-tRNA synthetase measured in fibroblasts of two infantile and one adolescent form was: 6.1, 2.2, and 2.1 pmol of [(14)C]aminoacyl-tRNA formed/mug protein/10 min, respectively. In addition, the cysteine K(m)'s for the same cells, respectively, were: 3.1 muM, 1.5 muM, and 1.2 muM. The corresponding data for specific activities of two normal cell lines were 2.0 and 5.1 pmol [(14)C]aminoacyl tRNA formed/mug protein/10 min, with K(m)'s of 3.0 muM and 1.7 muM. These data indicate that cystinotic cells contain levels of tRNA(Cys) and Cys-tRNA synthetase comparable to normal cells. In addition, within the cystinotic cells, the relative level of the Cys-tRNA synthetase and tRNA(Cys) to those of leucine and alanine are comparable to normal cells. Finally, the K(m) of Cys-tRNA synthetase for ATP and tRNA is similar in normal and cystinotic cells.

Amino Acids↗
PubMed1988

The independent regulation of tRNA(iMet) and tRNA(Asn) synthesis during Friend cell erythroid differentiation.

In this report, we have compared the changes in the production of tRNA(iMet) (initiator tRNA(Met] and tRNA(Asn), which occur during erythroid differentiation in the Friend erythroleukemia cell. The relative steady-state concentration of these two tRNAs (relative to the total tRNA population) was measured by aminoacylation. The results show that while the relative steady-state concentration of tRNA(iMet) changes very little in the cytoplasmic tRNA population, the relative concentration of tRNA(Asn) decreases during the first two days of differentiation and then undergoes an increase. This difference in the behavior of these two tRNAs is also seen when their relative concentrations in newly synthesized tRNA is examined. When tRNA is labeled with tritiated uridine for 24 h in vivo prior to isolation, the hybridization of this labeled tRNA to filter-bound tRNA genes shows that the relative concentration of tRNA(iMet) in newly synthesized tRNA changes very little, while the relative concentration of newly synthesized tRNA(Asn) again decreases through the first 2 days of differentiation, and then undergoes a smaller increase. Thus, the production of these two tRNAs appears to be independently regulated. Independent regulation of synthesis is also observed when examining the production of these two tRNAs in isolated nuclei. During erythroid differentiation, the relative synthesis of tRNA(iMet) (relative to total nuclear RNA synthesis) remains constant, while the relative synthesis of tRNA(Asn) undergoes periodic increases and decreases in value.

Animals↗
PubMed2000

Evidence for unfolding of the single-stranded GCCA 3'-End of a tRNA on its aminoacyl-tRNA synthetase from a stacked helical to a foldback conformation.

The conformation of a tRNA in its initial contact with its cognate aminoacyl-tRNA synthetase was investigated with the Escherichia coli glutamyl-tRNA synthetase-tRNA(Glu) complex. Covalent complexes between the periodate-oxidized tRNA(Glu) and its synthetase were obtained. These complexes are specific since none were formed with any other oxidized E. coli tRNA. The three major residues cross-linked to the 3'-terminal adenosine of oxidized tRNA(Glu) are Lys115, Arg209, and Arg48. Modeling of the tRNA(Glu)-glutamyl-tRNA synthetase based on the known crystal structures of Thermus thermophilus GluRS and of the E. coli tRNA(Gln)-glutaminyl-tRNA synthetase complex shows that these three residues are located in the pocket that binds the acceptor stem, and that Lys115, located in a 26 residue loop closed by coordination to a zinc atom in the tRNA acceptor stem-binding domain, is the first contact point of the 3'-terminal adenosine of tRNA(Glu). In our model, we assume that the 3'-terminal GCCA single-stranded segment of tRNA(Glu) is helical and extends the stacking of the acceptor stem. This assumption is supported by the fact that the 3' CCA sequence of tRNA(Glu) is not readily circularized in the presence of T4 RNA ligase under conditions where several other tRNAs are circularized. The two other cross-linked sites are interpreted as the contact sites of the 3'-terminal ribose on the enzyme during the unfolding and movement of the 3'-terminal GCCA segment to position the acceptor ribose in the catalytic site for aminoacylation.

Amino Acid Sequence↗
PubMed1994

Selenocysteine tRNA and serine tRNA are aminoacylated by the same synthetase, but may manifest different identities with respect to the long extra arm.

Selenocysteine (Sec) tRNA([Ser])Sec donates Sec to protein, but interestingly, this amino acid is synthesized on tRNA which is first aminoacylated with serine. Thus, the identity elements in tRNA([Ser])Sec for aminoacylation correspond to elements for seryl-tRNA synthetase recognition. As tRNA([Ser])Sec has low homology to the tRNA(Ser) isoacceptors, it would seem then that the identity elements in tRNA([Ser])Sec involve (1) very specific sequences, (2) conformational features, and/or (3) different points or domains for tRNA[Ser]Sec:synthetase and tRNASer:synthetase recognition. Initially, we confirmed that the same synthetase aminoacylates both tRNAs by showing that a mutant tRNA[Ser]Sec which has a blocked 3'-terminus is a competitive inhibitor of tRNASer aminoacylation with a partially purified and a highly purified seryl-tRNA synthetase preparation. The discriminator base (base G73) is essential for aminoacylation of tRNA([Ser])Sec and tRNA(Ser), while the long extra arm plays an important role which seems to be orientation- and length-specific in tRNA(Ser) and, in addition, may manifest sequence specificity in tRNA([Ser])Sec. This difference in the tRNA recognition specificity is discussed. The acceptor stem, DHU stem, and T phi C stem contribute to the recognition process, but to a lesser extent than the discriminator base and the long extra arm.

Animals↗
PubMed1994

Escherichia coli seryl-tRNA synthetase recognizes tRNA(Ser) by its characteristic tertiary structure.

To investigate the sequence requirements of Escherichia coli tRNA(Ser) for recognition by seryl-tRNA synthetase, various mutants of unmodified tRNA(Ser) were constructed. Substitution of G2.C71 by C2.G71, but not by A2.U71 or U2.A71, impaired the serine-accepting activity, indicating that this position is not involved in recognition by seryl-tRNA synthetase, but contributes to discrimination from other tRNAs processing C2.G71 such as tRNA(Leu). Other nucleotides characteristic of tRNA(Ser), including the discriminator base, were not involved in recognition by seryl-tRNA synthetase. The anticodon was not involved, as suggested by its sequence variety within the isoacceptors. The long variable arm composed of over ten nucleotides, which is a characteristic feature of tRNA(Ser) together with tRNA(Leu) and tRNA(Tyr), was stem-length-specifically, but not sequence-specifically, important for recognition. In order to introduce a sufficient serine-accepting activity to a tRNA(1LEU) transcript in vitro, besides the change from C2.G71 to G2.C71, the following elements had to be changed to those characteristic of tRNA(Ser): the sequence in the D-loop, the stem pairing pattern of the variable arm, the tertiary base-pair 15.48 and the nucleotide at position 59 in the T psi C-loop. None of the nucleotides at these changed positions was involved in base-specific recognition, indicating that seryl-tRNA synthetase selectively recognizes tRNA(Ser) on the basis of its characteristic tertiary structure rather than the nucleotides specific to tRNA(Ser).

Base Sequence↗
PubMed2001

Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria.

Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are (i) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and (ii) an aminoacyl-tRNA synthetase that aminoacylates the suppressor tRNA but no other tRNA in the cell. Here we describe two such aaRS-suppressor tRNA pairs, one for use in the yeast Saccharomyces cerevisiae and another for use in Escherichia coli. The "21st synthetase-tRNA pairs" include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppressor derived from E. coli initiator tRNA, for use in E. coli. The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs, and the aminoacylated tRNAs function efficiently in suppression of amber codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast. However, plasmids carrying the yeast TyrRS gene could not be stably maintained in E. coli. This lack of stability is most likely due to the fact that the wild-type yeast TyrRS misaminoacylates the E. coli proline tRNA. By using error-prone PCR, we have isolated and characterized three mutants of yeast TyrRS, which can be stably expressed in E. coli. These mutants still aminoacylate the suppressor tRNA essentially quantitatively in vivo but show increased discrimination in vitro for the suppressor tRNA over the E. coli proline tRNA by factors of 2.2- to 6.8-fold.

Amino Acids↗
PubMed1990

Evidence for two types of complexes formed by yeast tyrosyl-tRNA synthetase with cognate and non-cognate tRNA. Effect of ribonucleoside triphosphates.

Polyacrylamide gel electrophoresis at pH 8.3 was used to detect and quantitate the formation of the yeast tyrosyl-tRNA synthetase (an alpha 2-type enzyme) complex with its cognate tRNA. Electrophoretic mobility of the complex is intermediate between the free enzyme and free tRNA; picomolar quantities can be readily detected by silver staining and quantitated by densitometry of autoradiograms when [32P]tRNA is used. Two kinds of complexes of Tyr-tRNA synthetase with yeast tRNA(Tyr) were detected. A slower-moving complex is formed at ratios of tRNA(Tyr)/enzyme less than or equal to 0.5; it is assigned the composition tRNA.(alpha 2)2. At higher ratios, a faster-moving complex is formed, approaching saturation at tRNA(Tyr)/enzyme = 1; any excess of tRNA(Tyr) remains unbound. This complex is assigned the composition tRNA.alpha 2. The slower, i.e. tRNA.(alpha 2)2 complex, but not the faster complex, can be formed even with non-cognate tRNAs. Competition experiments show that the affinity of the enzyme towards tRNA(Tyr) is at least 10-fold higher than that for the non-cognate tRNAs. ATP and GTP affect the electrophoretic mobility of the enzyme and prevent the formation of tRNA.(alpha 2)2 complexes both with cognate and non-cognate tRNAs, while neither tyrosine, as the third substrate of Tyr tRNA synthetase, nor AMP, AMP/PPi, or spermidine, have such effects. Hence, the ATP-mediated formation of the alpha 2 structure parallels the increase in specificity of the enzyme towards its cognate tRNA.

Adenosine Monophosphate↗
PubMed2003

Recognition of acceptor-stem structure of tRNA(Asp) by Escherichia coli aspartyl-tRNA synthetase.

Protein-RNA recognition between aminoacyl-tRNA synthetases and tRNA is highly specific and essential for cell viability. We investigated the structure-function relationships involved in the interaction of the Escherichia coli tRNA(Asp) acceptor stem with aspartyl-tRNA synthetase. The goal was to isolate functionally active mutants and interpret them in terms of the crystal structure of the synthetase-tRNA(Asp) complex. Mutants were derived from Saccharomyces cerevisiae tRNA(Asp), which is inactive with E. coli aspartyl-tRNA synthetase, allowing a genetic selection of active tRNAs in a tRNA(Asp) knockout strain of E. coli. The mutants were obtained by directed mutagenesis or library selections that targeted the acceptor stem of the yeast tRNA(Asp) gene. The mutants provide a rich source of tRNA(Asp) sequences, which show that the sequence of the acceptor stem can be extensively altered while allowing the tRNA to retain substantial aminoacylation and cell-growth functions. The predominance of tRNA backbone-mediated interactions observed between the synthetase and the acceptor stem of the tRNA in the crystal and the mutability of the acceptor stem suggest that many of the corresponding wild-type bases are replaceable by alternative sequences, so long as they preserve the initial backbone structure of the tRNA. Backbone interactions emerge as an important functional component of the tRNA-synthetase interaction.

Aspartate-tRNA Ligase↗