Enzymatic hydrolysis of N-substituted aminoacyl-tRNA.

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 aminoacyltRNA'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-acetylleucyltRNA 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