Stabilization of aminoacyl-tRNA synthetases by sephadex and polyacrylamide gels (original) (raw)
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Enzymatic hydrolysis of N-substituted aminoacyl-tRNA
Proceedings of the National Academy of Sciences of the United States of America, 1967
The mechanism of the release of polypeptide chains from the ribosome-messenger RNA complex is not fully understood. It has been reported that free polypeptide chains are formed in cell-free protein-synthesizing systems, directed by polyribonucleotides, only if these polynucleotides contain statistically high frequencies of UAA codons.'-5 However, we do not know how the chain is released from the tRNA ribosome-mRNA complex after interruption of the translation by the UAA triplet. This release implies a hydrolysis of the ester bond between polypeptide and tRNA which could be catalyzed by a specific enzyme. The search for such an enzymatic activity necessitates the use of the relatively unstable polypeptidyl-tRNA's. It is difficult and laborious to prepare them in measurable quantities. In contrast, the chemically N-substituted aminoacyl-tRNA's, although having similar characteristics in other respects, are stable and readily synthesized.6' I An enzyme capable of hydrolyzing this ester linkage between N-acetylamino-acids and tRNA's has now been found in extracts of Escherichia coli. This enzyme was partially purified and several of its characteristics were studied. The enzyme also catalyzes the hydrolysis of di-phenylalanyl-tRNA and N-substituted oligopeptidyl-tRNA's. Material.-C'4-amino acids were obtained from the Commissariat a l'Energie Atomique (France); E. coli B tRNA, from General Biochemicals; crystalline pancreatic DNase and RNase, from Mann Research Laboratories; snake venom phosphodiesterase, from British Drug Houses Ltd.; T1 RNase, from Sigma Corp. E. coli leucine-specific tRNA of about 50% purity was a gift from Dr. M. Yaniv; and a sample of H3-diphenylalanyl tRNA, from Dr. C. Ganoza. The tRNA was charged with different C"4-amino acids in the presence of an E. coli 105,000 X g supernatant. The C'4-aminoacyl-tRNA was acetylated with acetic anhydride, as described by Haenni and Chapeville.7 In all cases it was shown that after acetylation all amino groups of the tRNA-bound amino acids were substituted. When serine and threonine are used it is possible that the OH groups also react with acetic anhydride, forming the corresponding esters. C'4-diphenylalanyl-tRNA was prepared according to Nakamoto and Kolakofsky8 by incubating C14-phenylalanyl-tRNA in the presence of ribosomes and 105,000 X g supernatant without addition of GTP. C'4-polylysyl-tRNA was prepared from an incubation mixture of E. coli ribosomes with C'4-lysyl-tRNA, poly A, GTP, and E. coli supernatant. Methods.-Analysis of the degradation products of N-acetylaminoacyl-tRNA: For most of the N-acetylaminoacyl-tRNA's, the method described below for N-acetylleucyl-tRNA was used. N-acetylleucine, leucine, N-acetylleucyladenosine (obtained after digestion of N-acetylleucyl-tRNA with pancreatic ribonuclease), and N-acetylleucyl-tRNA were separated by paper electrophoresis (Fig. 1). Under the same conditions, after treatment with RNase T1, two N-acetylleucyloligonucleotides were separated, one of which migrates with N-acetylleucine (Fig. 6). If a similar mixture had to be analyzed, both N-acetylleucyloligonucleotides would be converted to N-acetylleucyladenosine by treatment with pancreatic RNase before electrophoresis. N-acetylleucyl-tRNA, N-acetylleucyladenylate (N-acetylleucyl AMP, obtained after digestion with purified venom phosphodiesterase of N-acetylleucyl-tRNA), N-acetylleucyladenosine, and 2079
Methods, 2008
Here we describe the many applications of acid urea polyacrylamide gel electrophoresis (acid urea PAGE) followed by Northern blot analysis to studies of tRNAs. Acid urea PAGE allows the electrophoretic separation of different forms of a tRNA, discriminated by changes in bulk, charge, and/or conformation that are brought about by aminoacylation, formylation, or modification of a tRNA. Among the examples described are (i) analysis of the effect of mutations in the Escherichia coli initiator tRNA on its aminoacylation and formylation; (ii) evidence of orthogonality of suppressor tRNAs in mammalian cells and yeast; (iii) analysis of aminoacylation specificity of an archaeal prolyl-tRNA synthetase that can aminoacylate archaeal tRNA Pro with cysteine, but does not aminoacylate archaeal tRNA Cys with cysteine; (iv) identification and characterization of the AUAdecoding minor tRNA Ile in archaea; and (v) evidence that the archaeal minor tRNA Ile contains a modified base in the wobble position different from lysidine found in the corresponding eubacterial tRNA.
European journal of biochemistry / FEBS, 1976
By following the tryptophan fluorescence of yeast seryl-tRNA synthetase on addition of tRNA Ser it was observed that the number of binding sites for tRNA decreases from two to one with increasing temperature, ATP or KCl concentration. Concomitantly a considerable decrease of the apparent binding constant was observed. The variation in the number of binding sites is explained by the presence of at least one temperature and ionic strength sensitive binding site and one temperature and ionic strength independent binding site. Relaxation kinetic experiments revealed two binding processes: a fast one depending on tRNA concentration and ionic strength and a slow one, which appeared to be independent of tRNA concentration and ionic strength. Enzyme kinetic studies showed that the activity of seryl-tRNA synthetase strongly depends on the KCl concentration and exhibits a maximum at 0.2 M KCl. Based on the data from relaxation and enzyme kinetic experiments a model is suggested for the recogn...
The activation of amino acid analogues by phenylalanyl- and tyrosyl-tRNA synthetases from plants
Phytochemistry, 1975
of Phe-and Tyr-tRNA synthetases were obtained from seed or seedlings of Phaseolus aureus, Delonix regia and Caesalpinia tinctoria, and the ability of a variety of structural analogues of Phe or Tyr to act as alternative substrates or inhibitors was tested. 3-Hydroxymethylphenylalanine, a natural product of C. tinctoriu, formed a particularly effective substrate for the Tyr-tRNA synthetase from P. aureus. The structural features commensurate with substrate activity in an analogue molecule are discussed.
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.