Comparison of the Hydrolysis Patterns of Several tRNAs by Cobra Venom Ribonuclease in Different Steps of the Aminoacylation Reaction

Abstract

The hydrolysis of several tRNAs by an endonuclease extracted from the venom of Nuju oxiuna and specific for double-stranded, or at least highly ordered, regions has been studied under various experimental conditions.It is shown that the hydrolysis patterns of yeast tRNAPhe, tRNAval and tRNAAFP in the isolated state are similar, most of the cuts occurring in the anticodon and acceptor stems. Ionic conditions are able to modify the hydrolysis pattern. The origin of these modifications is discussed.The protection against ribonuclease action, afforded to tRNAPh", tRNA""' and tRNA"'P by the cognate aminoacyl-tRNA synthetase, is analyzed. It is shown that in all cases the anticodon stem is protected. The 3'-terminal region does not seem to be tightly engaged in the complex with the aminoacyl-tRNA synthetase. These results are discussed in the light of information on contact areas previously obtained by ultraviolet cross-linking techniques.The effects of the small ligands (ATP and amino acid) on the protection afforded to the tRNA by the cognate synthetase, have been studied. In the valine and aspartic acid systems, ATP induced a modification of the tRNAenzyme complex leading to differences in the hydrolysis pattern of the 3'-accepting region.The effects of aminoacylation on the cleavage of tRNAPhe, tRNA""' and tRNAA'p were also studied. Whereas no modification of the cleavage map was observed in the aspartic system, aminoacylation resulted in slight but significant modifications of the hydrolysis pattern for tRNAPh" and tRNAva' in the 3'-terminal region.Aminoacylation is one of the first steps of protein biosynthesis. This reaction includes several components (tRNAs, aminoacyl-tRNA synthetases, ATP, amino acids) and consists in several steps, the mechanisms of which have been intensively studied [l]. One of the most interesting questions is to know whether the tRNA molecules undergo conformational changes in the course of the aminoacylation process. Indeed, recent results showed such structural modifications, upon binding to the cognate aminoacyl-tRNA synthetase, in the yeast phenylalanyl system [2,3]. These changes may be responsible for the appearance of the catalytic activity or even for the specificity of aminoacylation [3]. Furthermorc, it has been shown that small ligands of aminoacyl-tRNA synthetases were able to alter the relations between tRNAPhe and phenylalanyl-tRNA synthetase, leading to asymmetric behaviour of the enzyme [4].The conformation of the aminoacyl-tRNA itself has been extensively studied and very conflicting results were reportcd in the litcrature concerning structure differences between aminoacylated and non-acylated tRNAs. In the last few years, however, several papers reported structural modifications of tRNA upon aminoacylation [5 -101.Finally, it is quite conceivable that cations may trigger or contribute to conformational changes in the isolated or ~~ ~

The tRNA-like structure of turnip yellow mosaic virus RNA: structural organization of the last 159 nucleotides from the 3′ OH terminus

“…This behaviour resembles that observed in several tRNAs where the D and Ti,t loops are inaccessible and the anticodon loops are readily accessible to nuclease SI (e.g., Wrede et al, 1979aWrede et al, , 1979bLockard and Kumar, 1981). In the corresponding stem regions, the strongest cuts by cobra venom RNase are in stem III, similar to the tRNAs where the anticodon stem is the most easily split by this enzyme (e.g., Favorova et al, 1981;Lockard and Kumar, 1981;Butorin et al, 1982). These results suggest that the three-dimensional structure of arms I1, III, and IV is similar to that of the corresponding parts in tRNA.…”

“…Limited enzymatic digestions were used to probe the structure of the last 159 nucleotides at the 3' OH terminus of TYMV RNA. This approach has already been used to establish the secondary structures of several small RNAs (e.g., Branlant et al, 1981;Toots et al, 1981) and to check structural features in tRNAs and rRNA (e.g., Wrede et al, 1979aWrede et al, , 1979bFavorova et al, 1981;Lockard and Kumar, 1981;Vassilenko et al, 1981;Butorin et al, 1982). The method depends on the differential accessibility of some residues or domains to nucleases.…”

History of tRNA research in strasbourg

“…Probing tRNA phosphate groups by ENU alkylation was pioneered and validated in studies on the tertiary structure of tRNA Phe and on protected phosphate groups in tRNA Val interacting with ValRS . Likewise, enzymatic probing with cobra venom nuclease specific of structured RNA domains was developed . In the case of tRNA:aaRS complexes, neutron scattering was of particular interest and was first essayed with yeast ValRS interacting with cognate or noncognate tRNAs .…”

TRNA and the guanosine triphosphatase activity of elongation factor Tu

“…The interaction between EF-Tu-GTP and aa-tRNA, leading to codon-directed binding of aa-tRNA to the ribosomal A-site, has been considered to be involved in the above-described effects Shorey et al, 1971; Kaziro, 1978). This interaction concerns multiple points of contact: besides the 3'-end (see above; Jonák et al, 1979Jonák et al, ,1980Jekowsky et al, 1977;Kruse et al, 1980;Pingoud et al, 1977), the aminoacyl-acceptor arm and the TtPC-stem also interact with the factor (see above; Boutorin et al, 1981Boutorin et al, , 1982Wikman et al, 1982). Recently, EF-Tu has been suggested to harbor an additional binding site for a second tRNA molecule (Van Noort et al, 1982.…”