Recognition by T factor of a tRNAphe yeast molecule recombined from 3′and 5′ halves; and its non messenger-dependent binding to ribosomes (original) (raw)
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Proceedings of the National Academy of Sciences, 1972
The synthesis of isoleucyl-tRNAPhe (Escherichia coli) proceeds at an appreciable rate under normal in vitro conditions in the presence of isoleucyl-tRNA synthetase (EC 6.1.1.5) from E. coli. The misacylated product is shown here to be hydrolyzed by highly purified phenylalanyl-tRNA synthetase from E. coli, with release of isoleucine and active tRNAPhe. Thus, phenylalanyl-tRNA synthetase possesses a previously unrecognized activity, which deacylates a mistakenly acylated tRNAPhe; the enzyme is inactive toward correctly matched aminoacyl tRNAs. Such a mechanism could serve to verify aminoacyl-tRNAs, deacylating those that are misacylated. Thts, a common generalization needs to be modified: an amino acid is not necessarily committed to a given (incorrect) anticodon when it is incorporated into aminoacyl-tRNA. It may be possible to correct it thereafter.
Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNAPhe
Biochemistry, 1987
Periodate-oxidized tRNAPhe (t RNA: :) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the az& enzyme with tRNA!: results in the loss of tRNAPhe aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-[ ''C]tRNA!: covalent complex indicates that the large (a , M , 87K) subunit of the enzyme interacts with the 3'-adenosine of t RNA: :. The [14C]tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography. The radioactivity was almost equally distributed among three peptides: Met-Lys[Ado]-Phe, Ala-Asp-Lys[Ado]-Leu, and Lys-Ile-Lys[Ado]-Ala. These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the a subunit. It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys). The latter sequence was just predicted to resemble the proposed consensus t R N A CCA binding region ' Abbreviations: Lys[Ado], N'-adenosyllysine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RPLC, reverse-phase high-performance liquid chromatography; PP,, inorganic pyrophosphate; RNase A, ribonuclease A. Aminoacyl-tRNA synthetases are abbreviated as a three-letter code of their specific amino acid followed by RS; the oneand three-letter amino acid codes are those suggested by IUPAB-IUB Commission on Biochemical Nomenclature.
Equivalent and Nonequivalent Binding Sites for tRNA on Aminoacyl-tRNA Synthetases
European Journal of Biochemistry, 1975
Complexes between tRNAPhe (yeast), tRNAser (yeast) and tRNATy' (Escherichia coli) and their cognate aminoacyl-tRNA synthetases have been studied by sedimentation velocity runs in an analytical ultracentrifuge. The amount of complex formation was determined by the absorption and the sedimentation coefficients of the fast-moving boundary in the presence of excess tRNA or excess synthetase respectively. The same method has been applied to unspecific combinations of tRNAs and synthetases. Inactive material of tRNA or synthetase does not influence the results.
Promoting the Formation of an Active Synthetase/tRNA Complex by a Nonspecific tRNA-binding Domain
Journal of Biological Chemistry, 2008
Previous studies showed that valyl-tRNA synthetase of Saccharomyces cerevisiae contains an N-terminal polypeptide extension of 97 residues, which is absent from its bacterial relatives, but is conserved in its mammalian homologues. We showed herein that this appended domain and its human counterpart are both nonspecific tRNA-binding domains (K d ϳ 0.5 M). Deletion of the appended domain from the yeast enzyme severely impaired its tRNA binding, aminoacylation, and complementation activities. This N-domain-deleted yeast valyl-tRNA synthetase mutant could be rescued by fusion of the equivalent domain from its human homologue. Moreover, fusion of the N-domain of the yeast enzyme or its human counterpart to Escherichia coli glutaminyl-tRNA synthetase enabled the otherwise "inactive" prokaryotic enzyme to function as a yeast enzyme in vivo. Different from the native yeast enzyme, which showed different affinities toward mixed tRNA populations, the fusion enzyme exhibited similar binding affinities for all yeast tRNAs. These results not only underscore the significance of nonspecific tRNA binding in aminoacylation, but also provide insights into the mechanism of the formation of aminoacyl-tRNAs. Aminoacyl-tRNA synthetases are a group of ancient enzymes, each of which catalyzes the attachment of a specific amino acid to its cognate tRNAs. Aminoacyl-tRNAs are then delivered by elongation factor-1 (EF-1) 3 to ribosomes for protein translation. In prokaryotes, there are typically 20 aminoacyl-tRNA synthetases, one for each amino acid (1-4). In eukaryotes, protein synthesis occurs not only in the cytoplasm, but also in organelles, such as mitochondria and chloroplasts (5). Thus, eukaryotes, such as yeast, commonly have two genes that encode distinct sets of proteins for each aminoacylation activity, one localized to the cytoplasm and the other to the mitochondria. Each set aminoacylates the isoaccepting tRNAs