Partial digestion of tRNA-aminoacyl-tRNA synthetase complexes with cobra venom ribonuclease

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Abstract
Transfer RNA [tRNA] molecules were labeled with 32P at the 5'' or 3'' end and digested with cobra venom RNase, which cut double-stranded regions. The products of yeast tRNAPhe and tRNAVal were analyzed by high-resolution gel electrophoresis. In the free state, tRNA were cut predominantly in the acceptor and anticodon stems. Minor cuts occurred in the T.PSI. stem in tRNAVal. The topography of zones interacting with their cognate synthetases was studied by determining tRNA regions shielded by protein. Nearly 100% protection was found in anticodon and acceptor stem of tRNAVal. In tRNAPhe, only the stem of the anticodon was protected. Noncognate interactions between tRNAPhe and tryptophanyl-tRNA synthetase from beef pancreas were examined. Beef enzyme did not protect tRNAPhe despite the fact that efficient misaminoacylation occurred. The pattern of shielding obtained for each tRNA-synthetase complex was compared with results of direct UV cross-linking experiments with these complexes.