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PubMed1980

The complex formation between Escherichia coli aminoacyl-tRNA, elongation factor Tu and GTP. The effect of the side-chain of the amino acid linked to tRNA.

The interaction between Escherichia coli aminoacyl-tRNAs and elongation factor Tu (EF-Tu) x GTP was examined. Ternary complex formation with Phe-tRNAPhe and Lys-tRNALys was compared to that with the respective misaminoacylated Tyr-tRNAPhe and Phe-tRNALys. There was no pronounced difference in the efficiency of aminoacyl-tRNA x EF-Tu x GTP complex formation between Phe-tRNAPhe and Tyr-tRNAPhe. However, Phe-tRNALys was bound preferentially to EF-Tu x GTP as compared to Lys-tRNALys. This was shown by the ability of EF-Tu x GTP to prevent the hydrolysis of the aminoacyl ester linkage of the aminoacyl-tRNA species. Furthermore, gel filtration of ternary complexes revealed that the complex formed with the misaminoacylated tRNALys was also more stable than the one formed with the correctly aminoacylated tRNALys. Both misaminoacylated aminoacyl-tRNA species could participate in the ribosomal peptide elongation reaction. Poly(U)-directed synthesis of poly(Tyr) using Tyr-tRNAPhe occurred to a comparable extent as the synthesis of poly(Phe) with Phe-tRNAPhe. In the translation of poly(A) using native Lys-tRNALys, poly(Lys) reached a lower level than poly(Phe) when Phe-tRNALys was used. It was concluded that the side-chain of the amino acid linked to a tRNA affects the efficiency of the aminoacyl-tRNA x EF-Tu x GTP ternary complex formation.

Amino Acyl-tRNA Synthetases↗
PubMed2004

The zinc-binding site of a class I aminoacyl-tRNA synthetase is a SWIM domain that modulates amino acid binding via the tRNA acceptor arm.

In its tRNA acceptor end binding domain, the glutamyl-tRNA synthetase (GluRS) of Escherichia coli contains one atom of zinc that holds the extremities of a segment (Cys98-x-Cys100-x24-Cys125-x-His127) homologous to the Escherichia coli glutaminyl-tRNA synthetase (GlnRS) loop where a leucine residue stabilizes the peeled-back conformation of tRNAGln acceptor end. We report here that the GluRS zinc-binding region belongs to the novel SWIM domain family characterized by the signature C-x-C-xn-C-x-H (n = 6-25), and predicted to interact with DNA or proteins. In the presence of tRNAGlu, the GluRS C100Y variant has a lower affinity for l-glutamate than the wild-type enzyme, with Km and Kd values increased 12- and 20-fold, respectively. On the other hand, in the absence of tRNAGlu, glutamate binds with the same affinity to the C100Y variant and to wild-type GluRS. In the context of the close structural and mechanistic similarities between GluRS and GlnRS, these results indicate that the GluRS SWIM domain modulates glutamate binding to the active site via its interaction with the tRNAGlu acceptor arm. Phylogenetic analyses indicate that ancestral GluRSs had a strong zinc-binding site in their SWIM domain. Considering that all GluRSs require a cognate tRNA to activate glutamate, and that some of them have different or no putative zinc-binding residues in the corresponding positions, the properties of the C100Y variant suggest that the GluRS SWIM domains evolved to position correctly the tRNA acceptor end in the active site, thereby contributing to the formation of the glutamate binding site.

Amino Acid Sequence↗
PubMed1991

Arginyl-tRNA synthetase from Escherichia coli affinity labeling with 3'-oxidized tRNA(Arg).

The covalent modification of E. coli arginyl-tRNA synthetase by the 2',3'-dialdehyde derivative of tRNA(Arg) (tRNA(oxArg)) resulted in the complete inactivation of the ATP-PPi exchange and aminoacylation activities of the enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the ArgRS-tRNA(oxArg) covalent complexes indicated that two bands simultaneously appeared on the gel parallel with inactivation corresponding to different higher molecular weights. This result was different from that of the other aminoacyl-tRNA synthetase labeling systems as previously reported. Upon the ribonuclease treatment of the modified ArgRS, less than 15% of both the initial ATP-PPi exchange and aminocylation activities were recovered. During the whole process of labeling and RNase treatment, the two activities of the enzyme were closely associated.

Affinity Labels↗
PubMedDate not supplied

[A study of tRNA(adenine-1-)-methyltransferase from Thermus thermophilus HB8 with tRNA using enzymatic and spectral methods].

The contact sites of yeast tRNA1Val with tRNA(adenine-1-)-methyltransferase (EC 2.1.1.36) were studied by comparing the partial digests of free and enzyme bound tRNA. The RNAases Sa, V1, S1 and A were used for this purpose. Phosphodiester bonds on the proximal end of the acceptor stem and adjacent D- and T psi-stems, forming a continuous area, are protected by the methylase from the action of RNAases. On the contrary an enhancement of phosphodiester bond splitting of the D- and T psi-loops and of the anticodon stem is observed in the presence of methylase, which is interpreted as tertiary structure relaxation of tRNA owing to complex formation. The isotherm of ethidium bromide adsorption on free and methylase bound tRNA has shown that in enzyme shields approximately 50% of tRNA double stranded regions.

Binding Sites↗
PubMed1987

[Interaction of s4U8 region of tRNA Phe with tRNA-(adenine-1-)-methyltransferase from Thermus thermophilus].

Photoaffinity labelling of tRNA (adenine-1-)-methyltransferase with an E. coli tRNA(Phe) derivative bearing 4-azidophenylmercuro group attached to s4U residue as well as direct photocross-linking of the native tRNA(Phe) with the enzyme via s4U residue has been studied. Both techniques labelling gave similar results, leading to covalent attachment of tRNA(Phe) to the enzyme within a specific complex. The data obtained indicate unambigously that s4U residue contacts with tRNA (adenine-1-)-methyltransferase within the corresponding specific complex.

Cross-Linking Reagents↗
PubMedDate not supplied

[Study of the structure of tRNA by the energy migration method using fluorescent labels covalently bound to specific tRNA loci].

Optical and fluorescent characteristics of fluorescein covalently attached to 3'-end of tRNAFhe and X-nucleotide in the extra arm of several species of tRNA from E. coli have been studied. The probe is shown to be a sensitive factor indicating the conformational change of tRNA induced by Mg2+ and Na+ ions. By measuring the extent of energy transfer the distances between the fluorescent probe attached to 3'-terminus and X-nucleotide of tRNA and specific binding site of ethidium bromide on tRNA were determined to be 40.5 A and 32.5 A, respectively. The distances measured are in good agreement with the NMR spectroscopy data showing that the specific binding site for ethidium bromide on tRNA is localised near the sixth base pair of the acceptor stem.

Amino Acyl-tRNA Synthetases↗
PubMed1993

The recognition of E. coli glutamine tRNA by glutaminyl-tRNA synthetase.

A variety of genetic, biochemical and structural studies have been used to determine factors ensuring the accuracy of recognition by aminoacyl-tRNA synthetases for tRNA. The identity elements of Escherichia coli tRNA(Gln) are located mainly in the anticodon and acceptor stem, and ensure the accurate recognition of the tRNA by glutaminyl-tRNA synthetase. We summarize a number of experimental techniques to define the accuracy of aminoacylation in vivo and in vitro.

Amino Acyl-tRNA Synthetases↗
PubMed1994

[The role of the 3'-CCA sequence in the interaction of tRNA(Phe) from E. coli and Thermus thermophilus with homologous phenylalanyl-tRNA-synthetases].

The 3'-CCA end of tRNA(Phe) from E. coli and Thermus thermophilus was modified by stepwise degradation and ligation of the shortened tRNA with different trinucleotides (pUpUpA, (pA)3, (pC)3, (pU)3). Kinetic parameters for the aminoacylation reaction of modified tRNAs have been determined. The role of the 3'-terminal trinucleotide of tRNA(Phe) in tRNA binding and aminoacylation by phenylalanyl-tRNA synthetases from E. coli and Thermus thermophilus is postulated.

Acylation↗
PubMed1998

A site in the dinucleotide-fold domain contributes to the accuracy of tRNA selection by Escherichia coli methionyl-tRNA synthetase.

Interactions of specific amino acid residues of the carboxyl-terminal domain of MetRS with the CAU anticodon of tRNAMet assure accurate and efficient aminoacylation. The substitution of one such residue, Trp461 by Phe, impairs the binding of cognate tRNA, but enhances the binding of noncognate tRNAs, particularly those containing G at the wobble position. However, the enhanced binding of noncognate tRNAs is not accompanied by the increased aminoacylation of these tRNAs. A genetic screening procedure was designed to isolate methionyl-tRNA synthetase mutants which were able to aminoacylate a GGU (threonine) anticodon derivative of tRNAfMet. One such mutant, obtained from W461F MetRS, had an Ile29 to Thr substitution in helix A located in the amino-terminal dinucleotide-fold domain that forms the site for amino acid activation. Analysis of the catalytic properties of the I29T/W461F enzyme indicates that the mutation in helix A of the dinucleotide-fold domain affects kcat for aminoacylation of tRNAs having a GGU threonine anticodon. Interactions with cognate tRNAfMet (CAU), as well as with methionine and ATP were not affected by the Ile29 to Thr substitution. We conclude that the I29T substitution leads to a slight adjustment of the alignment of the CCA stem of noncognate tRNAs (GGU) in the catalytic domain of the enzyme, reflected in the increase in kcat, which also allows mischarging in vivo. A function of Ile29 is therefore to minimize the mischarging of tRNAThr (GGU) by methionyl-tRNA synthetase. The methods described here provide useful tools for examining the mechanisms of tRNA selection by aminoacyl-tRNA synthetases.

Anticodon↗
PubMed1988

Glycyl-tRNA synthetase of Escherichia coli: immunological homology with phenylalanyl-tRNA synthetase.

Antibodies to Escherichia coli glycyl-tRNA synthetase (GlyRS) cross-react extensively with E. coli phenylalanyl-tRNA synthetase (PheRS). These data indicate that structural homology exists between these two enzymes, the only two aminoacyl-tRNA synthetases in E. coli having an alpha 2 beta 2 subunit structure. Although only limited similarities are found in the protein sequences deduced from their known gene sequences, the presence of common epitopes in GlyRS and PheRS adds to a rather long list of physical and chemical similarities between those proteins. In addition, antibodies directed at the alpha- and beta-subunits of GlyRS inhibit both GlyRS and PheRS in the same relative manner, indicating that the function as well as the structure of subunits is similar in each enzyme. In contrast, GlyRS antibodies did not cross-react with a number of other aminoacyl-tRNA synthetase activities from E. coli, yeast, or Bacillus.

Amino Acyl-tRNA Synthetases↗
PubMed1990

Aspartyl-tRNA synthetase from Escherichia coli: cloning and characterisation of the gene, homologies of its translated amino acid sequence with asparaginyl- and lysyl-tRNA synthetases.

By screening of an Escherichia coli plasmidic library using antibodies against aspartyl-tRNA synthetase (AspRS) several clones were obtained containing aspS, the gene coding for AspRS. We report here the nucleotide sequence of aspS and the corresponding primary structure of the aspartyl-tRNA synthetase, a protein of 590 amino acid residues with a Mr 65,913, a value in close agreement with that observed for the purified protein. Primer extension analysis of the aspS mRNA using reverse transcriptase located its 5'-end at 94 nucleotides upstream of the translation initiation AUG; nuclease S1 analysis located the 3'-end at 126 nucleotides downstream of the stop codon UGA. Comparison of the DNA-derived protein sequence with known aminoacyl-tRNA sequences revealed important homologies with asparaginyl- and lysyl-tRNA synthetases from E.coli; more than 25% of their amino acid residues are identical, the homologies being distributed preferencially in the first part and the carboxy-terminal end of the molecule. Mutagenesis directed towards a consensus tetrapeptide (Gly-Leu-Asp-Arg) and the carboxy-terminal end showed that both domains could be implicated in catalysis as well as in ATP binding.

Amino Acid Sequence↗
PubMed1976

Phenylalanyl-tRNA and seryl-tRNA synthetases from baker's yeast. Substrate specificity with regard to ATP analogs and mechanism of the aminoacylation reaction.

Eighteen analogs of ATP have been tested in the aminoacylation reaction of phenylalanyl-tRNA and seryl-tRNA synthetases from baker's yeast. Four compounds are substrates for phenylalanyl-tRNA synthetase, five for seryl-tRNA synthetase, one compound is an inhibitor for both enzymes; their Km and Ki and V values have been determined. The substrate specificity shows that for the catalytic action of both enzymes with these substrates positions 6, 7, 8 and 9 of the purine moiety and positions 2' and 3' of the ribose moiety are important.

Adenosine Triphosphate↗
PubMed1983

Characterization of a homogeneous arginyl- and lysyl-tRNA synthetase complex isolated from rat liver. Kinetic mechanism for lysyl-tRNA synthetase.

Bisubstrate kinetics and end product and dead end inhibition studies were performed on lysyl-tRNA synthetase isolated from rat liver. The kinetic patterns obtained are consistent with a sequential ordered mechanism of substrate addition, tRNA bound first, followed by lysine, and then by ATP. Pyrophosphate and AMP are released in a random fashion with aminoacylated tRNA the last product to dissociate from the enzyme. This is the first report of a kinetic mechanism for lysyl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1994

tRNA structure and ribosomal function. II. Interaction between anticodon helix and other tRNA mutations.

Using multiply mutated tRNA genes, we have studied unusual coding by tRNAs that have altered nucleotides (nt) 27-43, which normally form the top base-pair of the anticodon helix. In vivo, nt 27-43 mutations accelerate non-canonical C-A coding at the third (3') codon position 14-fold, similar to the 40-fold stimulation originally shown for first (5') codon position non-canonical G-U pairing. Thus the effects of nt 27-43 generalize to a second type of unusual coding. Nt 27-43 changes have a similar relative effect on tRNA level, aminoacylation, and ribosomal activity, despite concurrent changes of the 3' anticodon nucleotide which alter coding. However, under conditions of efficient aminoacylation, only a fraction of these (potential missense) anticodon changes can be recovered, suggesting toxicity. Available data support the idea that the effects of nt 27-43 are not particular to one codon. A previously isolated D-arm mutation (G24A) has a similar coding effect, enhancing both first position G-U wobble up to 130-fold, the third position C-A mispairing 40-fold. Anticodon helix mutations at 27-43 have little effect on 3' or 5' miscoding in the presence of the G24A D-arm mutation, and reciprocally, the D-arm's effects are much diminished in the presence of the anticodon helix mutations. Because these two tRNA loci alter both types of aberrant coding, and because they are highly interdependent, they may exploit a similar mechanism, dependent on a similar effect on tRNA conformation. We suggest a relatively non-specific decrease in the ribosomal rejection rates for tRNAs altered at anticodon helix nucleotides 27 and 43. Thus coding via non-canonical pairings at both 5' and 3' ends of the codon-anticodon helix has a measurable rate in vivo. However, we find that normal tRNA structure minimizes the efficiency of this aberrant translation. To put these same findings in another light, tRNAs bearing identical anticodons, if altered in structure elsewhere, may translate the genetic code differently.

Anticodon↗
PubMed1990

Sequence analysis of three tRNA(Phe) nuclear genes and a mutated gene, and one gene for tRNA(Ala) from Arabidopsis thaliana.

Three genes and one mutant gene for tRNA(Phe) (GAA) and one gene for tRNA(Ala) (UGC) were isolated from a whole-cell DNA library of Arabidopsis thaliana. All three tRNA(Phe) genes are identical in their nucleotide sequence, but differ in their 5' and 3' flanking regions. The mutant tRNA(Phe) (GAA) gene differs from the other three genes by one nucleotide change from highly conserved G to C at the 57th nucleotide position. The primary structure of the first tRNA(Ala) gene was also determined in this experiment.

Base Sequence↗
PubMed1989

Duplication of the tRNA(MMet) and tRNA(Cys) genes and of fragments of a gene encoding a subunit of the NADH dehydrogenase complex in Neurospora grassa mitochondrial DNA.

Neurospora crassa mitochondrial DNA (mtDNA) contains duplications of the tRNA(MMet) gene upstream of a gene (ND2) encoding a subunit of the NADH dehydrogenase complex and of the tRNA(Cys) gene which is found downstream of the apocytochrome b gene. Both duplicated genes are located upstream of the small rRNA gene. The duplications are extended to flanking sequences. In the case of the tRNA(MMet) duplication, two fragments of the ND2 gene are also duplicated. These two fragments, which are not contiguous in the ND2 gene, are connected to each other by a palindromic sequence of 37 bp and together they constitute an open reading frame. The possible involvement of this palindromic sequence in the processes of gene duplication and transfer is discussed. Two overlapping reading frames are present between the tRNA(MMet) and tRNA(Cys) copies. All information of the ND2 duplication and the two overlapping reading frames are present on a polycistronic transcript.

Amino Acid Sequence↗
PubMed2000

Mitochondrial DNA of Hydra attenuata (Cnidaria): a sequence that includes an end of one linear molecule and the genes for l-rRNA, tRNA(f-Met), tRNA(Trp), COII, and ATPase8.

The 3231-nucleotide-pair (ntp) sequence of one end of one of the two linear mitochondrial (mt) DNA molecules of Hydra attenuata (phylum Cnidaria, class Hydrozoa, order Anthomedusae) has been determined. This segment contains complete genes for tRNA(f-Met), l-rRNA, tRNA(Trp), subunit 2 of cytochrome c oxidase (COII), subunit 8 of ATP synthetase (ATPase8), and the 5' 136 ntp of ATPase6. These genes are arranged in the order given and are transcribed from the same strand of the molecule. As in two other cnidarians, the hexacorallian anthozoan Metridium senile and the octocorallian anthozoan Sarcophyton glaucum, the mt-genetic code of H. attenuata is near standard. The only modification appears to be that TGA specifies tryptophan rather than termination. Also as in M. senile and S. glaucum, the encoded H. attenuata mt-tRNA(f-Met) has primary and secondary structural features resembling those of Escherichia coli initiator tRNA(t-Met). As the encoded mt-tRNA(Trp) cannot be folded into a totally orthodox secondary structure, two alternative forms are suggested. The encoded H. attenuata mt-l-rRNA is 1738 nt, which is 451 nt shorter than the M. senile mt-l-rRNA. Comparisons of secondary structure models of these two mt-l-rRNAs indicate that most of the size difference results from loss of nucleotides in the H. attenuata molecule at a minimum of 46 locations, which includes elimination of six distinct helical elements.

Adenosine Triphosphatases↗
PubMed1995

Structural analysis of the interaction of the tRNA modifying enzymes Tgt and QueA with a substrate tRNA.

The enzymes tRNA guanine-transglycosylase (Tgt) and S-adenosylmethionine :tRNA ribosyltransferase-isomerase (QueA) participate in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q) in Escherichia coli. Here we show by HPLC analysis and gel retardation that both enzymes interact with an in vitro transcribed tRNA(ASP) from yeast, specifically modified with a Q precursor molecule. RNase I footprinting experiments showed strong protein tRNA contacts in the anticodon stem-loop and a minor interaction with the dihydrouridine loop. This suggests that all identity elements for the recognition of Q-specific tRNAs are clustered in the anticodon region and explains earlier results that both enzymes accept a RNA microhelix with the sequence of an anticodon stem-loop as substrate.

Anticodon↗