Study of the interaction of yeast arginyl-tRNA synthetase with yeast tRNAArg2 and tRNAArg3 by partial digestions with cobra venom ribonuclease.

Abstract

Yeast tRNAArg2 and tRNAArg3 are two isoacceptors which show similar V and Km for yeast arginyl-tRNA synthetase despite important differences in their primary structures. Fragments resulting from the partial digestion of 3' or 5' end-labelled tRNAArg2 and tRNAArg3 in the presence or absence of arginyl-tRNA synthetase by cobra venom ribonuclease, an enzyme which cuts preferentially in double-stranded regions, were analysed by electrophoresis on polyacrylamide gels. In the absence of arginyl-tRNA synthetase, major cuts were observed in tRNAArg2 and tRNAArg3 at the end of the 3' part of the acceptor stem and in the 5' part of the anticodon stem, whereas the 5' part of the acceptor stem and the 3' part of the anticodon stem are only slightly cleaved. The D and the T stems are almost fully resistant to cobra venom ribonuclease attack confirming the strong tertiary structural organization of this region. In the presence of arginyl-tRNA synthetase the two or three last sites of the 3' halves of the acceptor stems and the sites in the 3' halves of the anticodon stems are almost completely protected against ribonuclease hydrolysis in both tRNAs; 31-69% protection of the sites located in the 5' halves of the anticodon stem is also observed. However, the cleavage levels are enhanced for the three head positions in the 3' halves of the acceptor stems and a new cut appears at the first position of this region in the case of tRNAArg3. The similarity of the protection patterns of tRNAArg2 and tRNAArg3 suggests that both molecules interact in nearly the same manner with arginyl-tRNA synthetase, which in turn implies great similarities in their tertiary structure when involved in the complex. If this tertiary organization is like that described for tRNAPhe, all protected sites are located in the inside of its L-shaped model.