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At least 595 records · Page 33Linked to original sources
PubMed2000

Structural basis for double-sieve discrimination of L-valine from L-isoleucine and L-threonine by the complex of tRNA(Val) and valyl-tRNA synthetase.

Valyl-tRNA synthetase (ValRS) strictly discriminates the cognate L-valine from the larger L-isoleucine and the isosteric L-threonine by the tRNA-dependent "double sieve" mechanism. In this study, we determined the 2.9 A crystal structure of a complex of Thermus thermophilus ValRS, tRNA(Val), and an analog of the Val-adenylate intermediate. The analog is bound in a pocket, where Pro(41) allows accommodation of the Val and Thr moieties but precludes the Ile moiety (the first sieve), on the aminoacylation domain. The editing domain, which hydrolyzes incorrectly synthesized Thr-tRNA(Val), is bound to the 3' adenosine of tRNA(Val). A contiguous pocket was found to accommodate the Thr moiety, but not the Val moiety (the second sieve). Furthermore, another Thr binding pocket for Thr-adenylate hydrolysis was suggested on the editing domain.

Adenosine↗
PubMed1999

The structure of threonyl-tRNA synthetase-tRNA(Thr) complex enlightens its repressor activity and reveals an essential zinc ion in the active site.

E. coli threonyl-tRNA synthetase (ThrRS) is a class II enzyme that represses the translation of its own mRNA. We report the crystal structure at 2.9 A resolution of the complex between tRNA(Thr) and ThrRS, whose structural features reveal novel strategies for providing specificity in tRNA selection. These include an amino-terminal domain containing a novel protein fold that makes minor groove contacts with the tRNA acceptor stem. The enzyme induces a large deformation of the anticodon loop, resulting in an interaction between two adjacent anticodon bases, which accounts for their prominent role in tRNA identity and translational regulation. A zinc ion found in the active site is implicated in amino acid recognition/discrimination.

Amino Acyl-tRNA Synthetases↗
PubMed2001

Determination of tRNA(Phe) nucleotides contacting the subunits of Thermus thermophilus phenylalanyl-tRNA synthetase by photoaffinity crosslinking.

The nucleotides of tRNA(Phe) interacting with the subunits of Thermus thermophilus phenylalanyl-tRNA synthetase (the alpha(2)beta(2) heterotetramer) have been determined by photoaffinity crosslinking of randomly s(4)U-monosubstituted tRNA(Phe) transcripts which retain aminoacylation parameters closely similar to those of the native tRNA(Phe). The thiolated transcripts have been fractionated by affinity electrophoresis and separately crosslinked to the enzyme. Sites of crosslinking to the beta subunit have been identified at positions 33 and 39 and crosslinking sites to the alpha subunit have been localized at positions 20, 45 and 47, using alkaline hydrolysis analysis of the crosslinked proteinase K-treated tRNAs. An additional crosslink to the beta subunit, not identified in the full-length crosslinked tRNAs, has been deduced to occur at position 12, based on the analysis of an unusual (fast migrating) crosslinked product. Nucleotide s(4)U8 of native tRNA(Phe) has been shown to form a minor crosslink to the alpha subunit. Four of the seven crosslinking sites, namely nucleotides 8, 12, 20 and 39, are among those shown to be protected against cleavage by iodine in footprinting experiments; in contrast, only nucleotide 12 is among the contact sites defined in the crystal structure. The data of independent biochemical approaches strongly suggest conformational flexibility of the complex under functional conditions, thus reflecting the importance of macromolecular dynamics for the interaction.

Binding Sites↗
PubMed1991

Interaction of the tRNA(Phe) acceptor end with the synthetase involves a sequence common to yeast and Escherichia coli phenylalanyl-tRNA synthetases.

Modified lysines resulting from the cross-linking of the 3' end of tRNA(Phe) to yeast phenylalanyl-tRNA synthetase (an enzyme with an alpha 2 beta 2 structure) have been characterized by sequencing the labeled chymotryptic peptides that were isolated by means of gel filtration and reversed-phase chromatography. The analysis showed that Lys131 and Lys436 in the alpha subunit are the target sites of periodate-oxidized tRNA(Phe). Mutant protein with a Lys----Asn substitution established that each lysine contributes to the binding of the tRNA but is not essential for catalysis. The major labeled lysine (K131) belongs to the sequence IALQDKL (residues 126-132), which shares three identities with the peptide sequence ADKL found around the tRNAox-labeled Lys61 in the large subunit of Escherichia coli phenylalanyl-tRNA synthetase [Hountondji, C., Schmitter, J. M., Beauvallet, C., & Blanquet, S. (1987) Biochemistry 26, 5433-5439].

Amino Acid Sequence↗
PubMed1978

Aminoacyl-tRNA synthetases from yeast: generality of chemical proofreading in the prevention of misaminoacylation of tRNA.

The specificity of valyl-, phenylalanyl-, and tyrosyl-tRNA synthetases from yeast has been examined by a series of stringent tests designed to eliminate the possibility of artefactual interference. Valyl-tRNA synthetase, as well as activating a number of amino acid analogues, will accept alanine, cysteine, isoleucine, and serine in addition to threonine as substrates for both ATP-PPi exchange and transfer to some tRNAVal species. The transfer is not observed if atempts are made to isolate the appropriate aminoacyl-tRNAVal-C-C-A but its role in the overall aminoacylation can be suspected from both the formation of a stable aminoacyl-tRNAVal-C-C-A(3'NH2) compound and from the stoichiometry of ATP hydrolysis during the aminoacylation of the native tRNA. Similar tests with phenylalanyl-tRNA synthetase indicate that this enzyme will also activate and transfer other naturally occurring amino acids, namely, leucine, methionine, and tyrosine. The tyrosine enzyme, which lacks the hydrolytic capacity of the other two enzymes (von der Haar, F., & Cramer, F (1976) Biochemistry 15, 4131--4138) is probably absolutely specific for tyrosine. It is concluded that chemical proofreading, in terms of an enzymatic hydrolysis of a misacylated tRNA, plays an important part in maintaining the specificity in the overall reaction and that this activity may be more widespread than has so far been suspected.

Amino Acids↗
PubMed2002

tRNA-like recognition of group I introns by a tyrosyl-tRNA synthetase.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active RNA structure. Previous work suggested that CYT-18 recognizes a conserved tRNA-like structure of the group I intron catalytic core. Here, directed hydroxyl-radical cleavage assays show that the nucleotide-binding fold and C-terminal domains of CYT-18 interact with the expected group I intron cognates of the aminoacyl-acceptor stem and D-anticodon arms, respectively. Further, three-dimensional graphic modeling, supported by biochemical data, shows that conserved regions of group I introns can be superimposed over interacting regions of the tRNA in a Thermus thermophilus TyrRS/tRNA(Tyr) cocrystal structure. Our results support the hypothesis that CYT-18 and other aminoacyl-tRNA synthetases interact with group I introns by recognizing conserved tRNA-like structural features of the intron RNAs.

Cloning, Molecular↗
PubMed1976

Subcellular localization of S-adenosyl-L-methionine:tRNA methyltransferases with aminoacyl-tRNA synthetases in human and mouse: normal and leukemic leukocytes.

The subcellular distributions of S-adenosyl-L-methionine:tRNA methyltransferases and aminoacyl-tRNA synthetases were investigated with the use of human and mouse normal and leukemic leukocyte cell lines. Differential centrifugation of homogenized cell suspensions produced three pelleted subcellular fractions (nuclear and membrane, microsomal, and postribosomal) and a supernatant fraction. Each fraction was assayed for both methyltransferase activity and synthetase activity. The largest amounts, 40-50%, of total methyltransferase and synthetase activities were localized in either the microsomal or the postribosomal fractions, depending on cell type. In addition, the highest specific activities of these two enzyme systems were found to be present in the microsomal and postribosomal fractions. The psotribosomal fraction from leukemic leukocytes had a methyltransferase specific activity higher than that of the microsomal fraction, while the same two fractions of normal leukocytes had approximately equal activities. Specific activities of aminoacyl-tRNA synthetases were found to be approximately equal for these two fractions, whether they were from normal or leukemic leukocytes. The activity of tRNA methyltransferases and synthetases within the postribosomal fraction of the cytoplasm suggests the existence of high-molecular-weight enzyme complexes for the modification as well as the aminoacylation of tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed2000

Catalytic peptide of human glutaminyl-tRNA synthetase is essential for its assembly to the aminoacyl-tRNA synthetase complex.

Human glutaminyl-tRNA synthetase (QRS) is one of several mammalian aminoacyl-tRNA synthetases (ARSs) that form a macromolecular protein complex. To understand the mechanism of QRS targeting to the multi-ARS complex, we analyzed both exogenous and endogenous QRSs by immunoprecipitation after overexpression of various Myc-tagged QRS mutants in human embryonic kidney 293 cells. Whereas a deletion mutant containing only the catalytic domain (QRS-C) was targeted to the multi-ARS complex, a mutant QRS containing only the N-terminal appended domain (QRS-N) was not. Deletion mapping showed that the ATP-binding Rossman fold was necessary for targeting of QRS to the multi-ARS complex. Furthermore, exogenous Myc-tagged QRS-C was co-immunoprecipitated with endogenous QRS. Since glutaminylation of tRNA was dramatically increased in cells transfected with the full-length QRS, but not with either QRS-C or QRS-N, both the QRS catalytic domain and the N-terminal appended domain were required for full aminoacylation activity. When QRS-C was overexpressed, arginyl-tRNA synthetase and p43 were released from the multi-ARS complex along with endogenous QRS, suggesting that the N-terminal appendix of QRS is required to keep arginyl-tRNA synthetase and p43 within the complex. Thus, the eukaryote-specific N-terminal appendix of QRS appears to stabilize the association of other components in the multi-ARS complex, whereas the C-terminal catalytic domain is necessary for QRS association with the multi-ARS complex.

Adenosine Triphosphate↗
PubMed2001

Divergent adaptation of tRNA recognition by Methanococcus jannaschii prolyl-tRNA synthetase.

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 tRNA(Pro) possesses prokaryotic features in the acceptor stem. We show here that, in some respects, recognition of tRNA(Pro) 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.

Amino Acid Sequence↗
PubMed2003

Aquifex aeolicus tRNA (Gm18) methyltransferase has unique substrate specificity. TRNA recognition mechanism of the enzyme.

Transfer RNA (guanosine-2')-methyltransferase (Gm-methylase) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to 2'-OH of G18 in the D-loop of tRNA. Based on their mode of tRNA recognition, Gm-methylases can be divided into the following two types: type I having broad specificity toward the substrate tRNA, and type II that methylates only limited tRNA species. Protein synthesized by in vitro cell-free translation revealed that Gm-methylase encoded in the Aquifex aeolicus genome is a novel type II enzyme. Experiments with chimeric tRNAs and mini- and micro-helix RNAs showed that the recognition region of this enzyme is included within the D-arm structure of tRNALeu and that a bulge is essentially required. Variants of tRNALeu, tRNASer, and tRNAPhe revealed that a combination of certain base pairs in the D-stem is strongly recognized by the enzyme, that 4 bp in the D-stem enhance methyl acceptance activity, and that the Py16Py17G18G19 sequence is important for efficient methyl transfer. The methyl acceptance activities of all the A. aeolicus tRNA genes, which can be classified into 14 categories on the basis of their D-arm structure, were tested. The results clearly showed that the substrate recognition mechanism elucidated by the variant experiments was applicable to their native substrates.

Amino Acid Sequence↗
PubMed1983

Effect of gamma radiation on E. coli ribosomes, tRNA and aminoacyl-tRNA synthetases.

Gamma-irradiated E coli ribosomes and tRNA, in aerated solutions, were inactivated with D37 doses of 144 and 77 Gy, respectively. Aminoacyl-tRNA-synthetases were only slightly inactivated under comparable conditions. Effects of additives to ribosome and tRNA solutions suggest that hydroxyl radicals were the major damaging species, that superoxide anions were not damaging and that radiolytically-formed hydrogen peroxide was also unimportant. Part of the damage by hydroxyl radicals is expressed through secondary radicals produced from additives and buffers. Results obtained with three different buffers suggest that (1) acetate ions provide protection by competing for hydroxyl radicals, (2) chloride ions are without effect and (3) inactivation of ribosomes and aminoacyl-tRNA-synthetases in Tris-HCl/MgCl2 and phosphate/MgCl2 buffered solutions was similar but the tRNA inactivation was lower in Tris-HCl/MgCl2 buffer.

Amino Acyl-tRNA Synthetases↗
PubMed1974

The role of the anticodon in the interaction between methionyl-tRNA synthetase and bacterial initiator tRNA.

Complementary and antiparallel oligonucleotides bind to exposed regions of the tRNA molecule. Aminoacylation in the presence of triplets has been used to determine the role of the anticodon in the interaction between methionyl-tRNA synthetase and initiator tRNA. ApUpG has no effect on the charging even when 70% of the tRNA is bound to the triplet, whereas in the presence of GpGpU which binds to the A-C-C sequence adjacent to the 3' terminal adenosine that fraction of the tRNA which is bound to the triplet is completely unavailable for charging. Hence the anticodon is probably not involved in a primary interaction while the A-C-C-A-OH clearly is. This conclusion is supported by the failure of the isolated anticodon loop and stem oligonucleotides to inhibit the aminoacylation reaction.

Amino Acyl-tRNA Synthetases↗
PubMed1975

Studies on human tRNA. I. The rapid, large scale isolation and partial fractionation of placenta and liver tRNA.

A procedure for the large scale isolation of mammalian tRNA has been applied to the isolation of several grams of human liver, human placenta, rabbit liver and rat liver tRNA. This procedure entails an initial grinding of the tissue in phenol-sodium acetate at acidic pH, followed by DEAE cellulose chromatography. Procedures are also described for analysis of the purified tRNA on the basis of size, using controlled pore glass bean columns. In addition, the acceptor activity of isolated tRNAs has been determined using both the heterologous and homologous synthetases. The chromatographic profile of individual isoaccepting species using BD cellulose chromatography is shown and the 3' terminal nucleoside content was also determined. The methods described now make it feasible for large scale studies of mammalian tRNA enabling us to better understand the relationships between the structure of mammalian tRNA and its many diversified functions.

Amino Acyl-tRNA Synthetases↗
PubMed1992

The T-loop region of animal mitochondrial tRNA(Ser)(AGY) is a main recognition site for homologous seryl-tRNA synthetase.

Recognition sites of bovine mitochondrial serine tRNA specific for condons AGY [tRNA(Ser) (AGY)] by the cognate mitochondrial seryl-tRNA synthetase were studied using a range of tRNA(Ser)(AGY) variants which were obtained by the in vitro transcription of synthetic tRNA genes with T7 RNA polymerase. Base replacements in the anticodon and discriminator sites did not affect serine acceptance. However, deletion and/or replacement in the T-loop region completely deprived the variants of their charging activities. Point mutation experiments in this region also showed that the adenosine residue in the middle of the T-loop (position 58), which is involved in tertiary interaction between the T-loop and the truncated D-arm [de Bruijn and Klug, 1983] played a significant role in the recognition process by the synthetase.

Animals↗
PubMed1995

Footprinting of tRNA(Phe) transcripts from Thermus thermophilus HB8 with the homologous phenylalanyl-tRNA synthetase reveals a novel mode of interaction.

The phosphates of the tRNA(Phe) transcript from Thermus thermophilus interacting with the cognate synthetase were determined by footprinting. Backbone bond protection against cleavage by iodine of the phosphorothioate-containing transcripts was found in the anticodon stem-loop, the D stem-loop and the acceptor stem and weak protection was also seen in the variable loop. Most of the protected phosphates correspond to regions around known identity elements of tRNA(Phe). Enhancement of cleavage at certain positions indicates bending of tRNAPhe upon binding to the enzyme. When applied to the three-dimensional model of tRNA(Phe) from yeast the majority of the protections occur on the D loop side of the molecule, revealing that phenylalanyl-tRNA synthetase has a rather complex and novel pattern of interaction with tRNAPhe, differing from that of other known class II aminoacyl-tRNA synthetases.

Base Composition↗
PubMed2003

Molecular recognition of proline tRNA by prolyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1.

To investigate the recognition mechanism of tRNA(Pro) by prolyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1, various tRNA(Pro) transcripts were prepared by in vitro transcription system. These transcripts were aminoacylated with proline by overexpressed A. pernix prolyl-tRNA synthetase. From prolylation experiments, recognition elements of A. pernix tRNA(Pro) were determined to be G35 and G36 of anticodon, discriminator base A73, and G1-C72 base pair at acceptor stem end.

Amino Acyl-tRNA Synthetases↗
PubMed2004

Determination of tryptophan tRNA recognition sites for tryptophanyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1.

To investigate the recognition mechanism of tryptophan tRNA by tryptophanyl-tRNA synthetase from extreme hyperthermophilic and aerobic archaeon, Aeropyrum pernix K1, tryptophanylation activities were examined by using mutant tryptophan tRNA transcripts prepared by in vitro transcription system. Their transcripts were aminoacylated with tryptophan by overexpressed A. pernix tryptophanyl-tRNA synthetase. The results indicated that anticodon nucleotides C34, C35 and A36, discriminator base A73, G1-C72 and G2-C71 base pairs of acceptor stem were base-specifically recognized by A. pernix tryptophanyl-tRNA synthetase.

Aeropyrum↗
PubMed2005

Recognition sites of glycine tRNA for glycyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1.

To elucidate the tRNA recognition sites of glycine tRNA from an extreme thermophilic and aerobic archaeon, Aeropyrum pernix K1, we examined glycylation activities using in vitro mutant glycine tRNA transcripts and recombinant A. pernix glycyl-tRNA synthetase. The recognition nucleotides were determined to be C35 and C36 of anticodon, C2-G71 and G3-C70 base-pairs of acceptor stem. However, discriminator base A73 was not recognized by glycyl-tRNA synthetase.

Aeropyrum↗