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PubMedDate not supplied

[Interaction between tRNA-recognizing sites of phenylalanyl-tRNA synthetase from Escherichia coli MRE-600].

Modification of phenylalanyl-tRNA synthetase by N-Br-Ac-[14C]Phe-tRNAPhe results in two products, one being stable in alkaline conditions and the other being instable. The instable product is formed in the presence of an equimolar excess of the reagent over the enzyme while the stable one--in the presence of a tenfold excess. The experimental results are described by a kinetic model which takes into account the existence of two routes of modification of product formation. The first route is from the complex containing one molecule of the reagent and the other from the complex containing two molecules of the reagent. It is assumed that the presence of the molecule of the reagent in one center of the dimeric enzyme induces a change in the acceptor point of the other center, which leads to local conformational changes of the enzyme molecule interacting with the substrate analogue.

Amino Acyl-tRNA Synthetases↗
PubMed2002

tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli.

We report the characterization of tadA, the first prokaryotic RNA editing enzyme to be identified. Escherichia coli tadA displays sequence similarity to the yeast tRNA deaminase subunit Tad2p. Recombinant tadA protein forms homodimers and is sufficient for site-specific inosine formation at the wobble position (position 34) of tRNA(Arg2), the only tRNA having this modification in prokaryotes. With the exception of yeast tRNA(Arg), no other eukaryotic tRNA substrates were found to be modified by tadA. How ever, an artificial yeast tRNA(Asp), which carries the anticodon loop of yeast tRNA(Arg), is bound and modified by tadA. Moreover, a tRNA(Arg2) minisubstrate containing the anticodon stem and loop is sufficient for specific deamination by tadA. We show that nucleotides at positions 33-36 are sufficient for inosine formation in mutant Arg2 minisubstrates. The anticodon is thus a major determinant for tadA substrate specificity. Finally, we show that tadA is an essential gene in E.coli, underscoring the critical function of inosine at the wobble position in prokaryotes.

Adenosine Deaminase↗
PubMed1975

Temporal appearance of bacteriophage T4-modified valyl tRNA synthetase in Escherichia coli.

Bacteriophage T4-induced modification of Escherichia coli vlayl-tRNA synthetase (EC 6.1.1.9) requires: synthesis of a phage-gene specified tau factor, addition of the factor to host valyl-tRNA synthetase to produce a urea-stable enzyme, and interaction of the modified enzyme with tRNA to produce a more rapidly sedimenting valyl-tRNA synthetase activity on sucrose density gradients. This report demonstrates that the coincident, chloramphenicol-sensitive appearance of urea-stable and rapidly sedimenting valyl-tRNA synthetase activity are immediate early phage functions. It implies that once the tau factor is synthesized, further interactions are stoichiometric rather than catalytic. The potential for valyl-tRNA synthetase modification accumylates when E. coli is infected with T4 PHAGE IN THE PRESENCE OF CHLORAMPHINICOL AND IS EXPRESSED DURING THE RESUMPTION OF PROTEIN SYNTHESIS WHEREAS FURTHER RNA synthesis is inhibited by rifampicin. The modification phenomenon occurs similarly in several strains of E. coli and represents a novel virus-host interaction.

Amino Acyl-tRNA Synthetases↗
PubMed1997

Chemical modifications of Bacillus subtilis tryptophanyl-tRNA synthetase.

A concerted conformational change in Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was evident from previous fluorescence on the quenching of the single Trp residue Trp-92 in the 4FTrp-AMP complexed enzyme. In this study, chemical modifications of the B. subtilis TrpRS were employed to further characterize this conformational change, with the single Trp residue serving as a marker for monitoring the change. Modifications of the enzyme by means of the Trp-specific agent N-bromosuccinimide (NBS) or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BNPS-skatole) inactivated the enzyme in accord with the essential role of Trp-92, as identified previously by site-directed mutagenesis. ATP sensitized TrpRS toward inactivation by NBS and BNPS-skatole, which suggested a conformational change that resulted in greater accessibility of Trp-92 toward modifications. In contrast, the cognate tRNATrp substrate exerted a specific protective effect against inactivation by both of the reagents, indicating that the TrpRS-tRNATrp interaction reduces the accessibility of Trp-92 under our experimental conditions. By comparison, modification of sulfhydryl groups by means of iodoacetamide did not reduce TrpRS activity. Observations on Trp-specific modification and substrate protection effects are discussed in the context of the Bacillus stearothermophilus TrpRS crystal structure.

Bacillus subtilis↗
PubMedDate not supplied

[Modifications of ribosomes from rat liver with alkylating derivatives of tRNA].

Alkylating analogs of peptidyl-tRNA: N-chloroambucilyl-14C-phenylanalyl-tRNA (1), N-iodoacetyl-14C-phenylalanyl-tRNA (2) and N-bromo-acetyl-14C-phenlalanyl-tRNA (3) were applied for the modification of the peptidyl-transferase center of the 80S ribosomes from rat liver. These analogs, being in the teronary complex poly-U: ribosome : tRNA analog, modified ribosomal proteins and ribosomal RNA. The modification is directed to large ribosomal subunit. It is found, that (1) modifies ribosomal proteins L5, L25, L31 and L32 and (2) modifies ribosomal proteins L4, L6, L10+L11, L13 and L30.

Animals↗
PubMed2001

Transfer RNA(Ala) recognizes transfer-messenger RNA with specificity; a functional complex prior to entering the ribosome?

tmRNA (SsrA or 10Sa RNA) functions as both a transfer RNA and a messenger RNA, rescues stalled ribosomes and clears the cell of incomplete polypeptides. We report that native Escherichia coli tmRNA interacts specifically with native or synthetic E.coli tRNA alanine (tRNA(Ala)) in vitro, alanine being the first codon of the tmRNA internal open reading frame. Aminoacylatable RNA microhelices also bind tmRNA. Complex formation was monitored by gel retardation assays combined with structural probes. Nucleotides from the acceptor stem of tRNA(Ala) are essential for complex formation with tmRNA. tRNA(Ala) isoacceptors recognize tmRNA with different affinities, with an important contribution from tRNA(Ala) post-transcriptional modifications. The most abundant tRNA(Ala) isoacceptor in vivo binds tmRNA with the highest affinity. A complex between tRNA(Ala) and tmRNA might involve up to 140 tmRNA molecules out of 500 present per E.coli cell. Our data suggest that tmRNA interacts with the tRNA that decodes the resume codon prior to entering the ribosome. Biological implications of promoting specific complexes between tmRNA and aminoacylatable RNAs are discussed, with emphasis on primitive versions of the translation apparatus.

Base Sequence↗
PubMed2005

molecular mechanism of lysidine synthesis that determines tRNA identity and codon recognition.

Lysidine (2-lysyl cytidine) is a lysine-containing cytidine derivative commonly found at the wobble position of bacterial AUA codon-specific tRNA(Ile). This modification determines both codon and amino acid specificities of tRNA(Ile). We previously identified tRNA(Ile)-lysidine synthetase (tilS) that synthesizes lysidine, for which it utilizes ATP and lysine as substrates. Here, we show that lysidine synthesis consists of two consecutive reactions that involve an adenylated tRNA intermediate. A mutation study revealed that Escherichia coli TilS discriminates tRNA(Ile) from the structurally similar tRNA(Met) having the same anticodon loop by recognizing the anticodon loop, the anticodon stem, and the acceptor stem. TilS was shown to bind to the anticodon region and 3' side of the acceptor stem, which cover the recognition sites. These findings reveal a dedicated mechanism embedded in tRNA(Ile) that controls its recognition and discrimination by TilS, and indicate the significance of this enzyme in the proper deciphering of genetic information.

Amino Acyl-tRNA Synthetases↗
PubMed1990

Existence of two forms of rat liver arginyl-tRNA synthetase suggests channeling of aminoacyl-tRNA for protein synthesis.

Arginyl-tRNA synthetase (arginine-tRNA ligase, EC 6.1.1.19) is found in extracts of mammalian cells both as a free protein (Mr = 60,000) and as a component (Mr approximately 72,000) of the high molecular weight aminoacyl-tRNA synthetase complex (Mr greater than 10(6). Several pieces of evidence indicate that the low molecular weight free form is not a proteolytic degradation product of the complex-bound enzyme but that it preexists in vivo: (i) the endogenous free form differs in size from the active proteolytic fragment generated in vitro, (ii) conditions expected to increase or decrease the amount of proteolysis do not alter the ratio of the two forms of the enzyme, and (iii) the free form contains an NH2-terminal methionine residue. A model is presented that provides a rationale for the existence of two forms of arginyl-tRNA synthetase in cells. In this model the complexed enzyme supplies arginyl-tRNA for protein synthesis, whereas the free enzyme provides arginyl-tRNA for the NH2-terminal arginine modification of proteins by arginyl-tRNA:protein arginyltransferase. This latter process targets certain proteins for removal by the ubiquitin-dependent protein degradation pathway. The necessity for an additional pool of arginyl-tRNA for the modification reaction leads to the conclusion that the arginyl-tRNA destined for protein synthesis (and/or protein modification) is channeled and unavailable for other processes. Other evidence supporting channeling in protein synthesis is discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Nucleoside modifications affect the structure and stability of the anticodon of tRNA(Lys,3).

NMR spectroscopy was used to determine the solution structures of RNA oligonucleotides comprising the anticodon domain of tRNA(Lys,3). The structural effects of the pseudouridine modification at position 39 were investigated and are well correlated with changes in thermodynamic parameters. The loop conformation differs from that seen in tRNA(Phe) and provides an explanation of the critical role of modification in this tRNA.

Anticodon↗
PubMed1994

Nuclear-encoded mitochondrial tRNAs of Trypanosoma brucei have a modified cytidine in the anticodon loop.

The mitochondrial genome of Trypanosoma brucei does not appear to encode any tRNA genes. Isolated organellar tRNAs hybridize to nuclear DNA, suggesting that they are synthesized in the nucleus and subsequently imported into the mitochondrion. Most imported tRNAs have cytosolic counterparts, showing identical mobility on two-dimensional polyacrylamide gels. We have compared three nuclear-encoded mitochondrial tRNAs (tRNA(Lys), tRNA(Leu), tRNA(Tyr)) with their cytosolic isoforms by direct enzymatic sequence analysis. Our findings indicate that the primary sequences of the mitochondrial and the corresponding cytosolic tRNAs are identical. However, we have identified a mitochondrion-specific nucleotide modification of each tRNA which is localized to a conserved cytidine residue at the penultimate position 5' of the anticodon. The modification present in mature mitochondrial tRNA(Tyr) was not found in a mutant tRNA(Tyr) defective in splicing in either cytosolic or mitochondrial fractions. The mutant tRNA(Tyr) has been expressed in transformed cells and its import into mitochondria has been demonstrated, suggesting that the modified cytidine residue is not required for import and therefore may be involved in adapting imported tRNAs to specific requirements of the mitochondrial translation machinery.

Animals↗
PubMed2026

tRNA methylation: functional insights and epitranscriptomic regulation.

tRNAs, one of the most conserved and abundant RNAs, are central components of protein synthesis, transferring genetic information from DNA to proteins through a precise base-pairing mechanism. Post-transcriptional modifications of tRNAs by tRNA modifying enzymes are essential for maintaining their normal physiological functions, including methylation, isomerization and glycosylation. tRNA methylation, particularly 1-methyladenosine (m1A), 5-methylcytidine (m5C), and 7-methylguanosine (m7G), are among the most abundant and diverse types of post-transcriptional modifications of tRNA, which promote the stability of tRNA secondary and tertiary structures and allow for proper translation. In addition, tRNA methylation affects the production and function of tsRNA (tRNA-derived small RNA), small fragments of RNA that further regulate gene expression and protein synthesis. In our review, we discuss the relevant biological functions of tRNA methylation, including tRNA stability, protein translation, and tsRNA biogenesis.

RNA, Transfer↗
PubMed2004

Phenotype of non-syndromic deafness associated with the mitochondrial A1555G mutation is modulated by mitochondrial RNA modifying enzymes MTO1 and GTPBP3.

Phenotypic expression of the deafness-associated mitochondrial A1555G mutation in the 12S rRNA gene is influenced by aminoglycosides and complex inheritance of nuclear-encoded modifier genes. The position of a major nuclear modifier gene has been localized to chromosome 8p23.1, but the identification of this gene has remained elusive. Recently, we identified a second modifier gene, mitochondrial transcription factor B1 (TFB1M), involved in mitochondrial rRNA modification. In the present study, we tested three genes involved in mitochondrial tRNA or rRNA modification, and two genes associated with non-syndromic deafness, for linkage and linkage disequilibrium (LD) in 214 DNA samples from Spanish, Italian, and Arab-Israeli families with maternally inherited non-syndromic hearing loss. The multipoint non-parametric linkage analysis and transmission disequilibrium test testing were done using all families combined as well as divided based on linkage to the chromosome 8 locus and ethnicity. Two genes, MTO1 and GTPBP3, showed strongly suggestive linkage and significant LD results. Since both genes, as well as TFB1M, are involved in the process of mitochondrial RNA modification, it appears that the modification of mitochondrial RNA is an important regulatory pathway in the phenotypic expression of the deafness-associated mitochondrial A1555G mutation. This conclusion was supported by comparing linkage results of simulated genotypes with actual results for the four genes involved in mitochondrial RNA modification.

Carrier Proteins↗
PubMed2005

Possible conformations of 5-aminomethyluridine derivatives recognizing a G at the third position of the codon.

Specificity of the codon-anticodon interaction is often modulated by post-transcriptional modification at the first position of the anticodon (position 34) of the tRNA molecules. The modification of U(34) into 5-aminomethyluridine derivatives facilitates the pairing of the base with a G at the third position of the codon (position III). It was proposed that this wobble pairing is dependent on the deprotonation of the uridine derivative [Takai and Yokoyama, Nucleic Acids Res., 31, 6383-6391 (2003)]. This seemed to explain many results from biochemical and genetic experiments. On the other hand, however, the geometry of the base pair between 5-methylaminomethyluridine and G(III) in a crystal of the ribosomal 30S subunit was quite different from the predicted one [Murphy IV et al., Nat. Struct. Mol. Biol., 11, 1186-1191 (2004)]. It is obvious that the deprotonation, if any, should be inefficient at the pH at which the crystal was made. Therefore, the crystal structure does not exclude the possibility that the pairs are primarily dependent on the deprotonation of the modified uridines at the physiological pH.

Anticodon↗
PubMed1994

Changes in translational accuracy of Escherichia coli when folate metabolism is perturbed.

Translational accuracy was monitored in Escherichia coli mutants which contain abnormal folate pools. A decrease in translational accuracy was indicated by the increased production of a T4 phage mutant containing a UGA mutation in a tail fibre gene. An E. coli folC mutant suppressed the phage mutant under conditions where it presumably accumulated methyl-tetrahydrofolate (methyl-THF). A UGA suppressor strain with the mutation affecting the primary structure of the tRNA(trp) normally suppressed the phage mutant. When the strain was made thymine-requiring it no longer suppressed. The accumulation of methyl-THF which permits suppression by thymine-requiring strains may act to interfere with suppression by the tRNA suppressor, possibly by changing the modification pattern of the tRNA.

Aminopterin↗
PubMed2004

A truncated aminoacyl-tRNA synthetase modifies RNA.

Aminoacyl-tRNA synthetases are modular enzymes composed of a central active site domain to which additional functional domains were appended in the course of evolution. Analysis of bacterial genome sequences revealed the presence of many shorter aminoacyl-tRNA synthetase paralogs. Here we report the characterization of a well conserved glutamyl-tRNA synthetase (GluRS) paralog (YadB in Escherichia coli) that is present in the genomes of >40 species of proteobacteria, cyanobacteria, and actinobacteria. The E. coli yadB gene encodes a truncated GluRS that lacks the C-terminal third of the protein and, consequently, the anticodon binding domain. Generation of a yadB disruption showed the gene to be dispensable for E. coli growth in rich and minimal media. Unlike GluRS, the YadB protein was able to activate glutamate in presence of ATP in a tRNA-independent fashion and to transfer glutamate onto tRNA(Asp). Neither tRNA(Glu) nor tRNA(Gln) were substrates. In contrast to canonical aminoacyl-tRNA, glutamate was not esterified to the 3'-terminal adenosine of tRNA(Asp). Instead, it was attached to the 2-amino-5-(4,5-dihydroxy-2-cyclopenten-1-yl) moiety of queuosine, the modified nucleoside occupying the first anticodon position of tRNA(Asp). Glutamyl-queuosine, like canonical Glu-tRNA, was hydrolyzed by mild alkaline treatment. Analysis of tRNA isolated under acidic conditions showed that this novel modification is present in normal E. coli tRNA; presumably it previously escaped detection as the standard conditions of tRNA isolation include an alkaline deacylation step that also causes hydrolysis of glutamyl-queuosine. Thus, this aminoacyl-tRNA synthetase fragment contributes to standard nucleotide modification of tRNA.

Adenosine↗