Complexes of Aminoacyl‐tRNA Synthetases with tRNAs as Studied by Partial Nuclease Digestion

Abstract

Complexes of tRNAPhe and tRNASer with their cognate aminoacyl tRNA synthetases have been exposed to a number of nucleases under conditions of partial digestion in order to gain information on the topography of the complexes. tRNAPhe was found to be strongly protected by yeast phenylalanyl tRNA synthetase at both nuclease‐sensitive sites, the dihydrouridine loop and the anticodon. tRNASer, on the other hand, was selectively shielded by yeast seryl tRNA synthetase at one of the two nuclease targets, the G‐C‐C‐A terminus, whereas splitting at the other target, the anticodon, was not affected at all by the synthetase.There was complete specificity of shielding: tRNAs were only protected by their respective synthetases. The complexes were sufficiently stable to allow an analysis of protection not only at pH 5.5 but also at pH 7.0. Phenylalanyl tRNA synthetase protected one mole of tRNAPhe, seryl tRNA synthetase two moles of tRNASer against nuclease attack. Denatured tRNAPhe did not bind to phenylalanyl tRNA synthetase.Two effects of ligands on complex stability have been observed. The analog phenylalaninol‐AMP increased the protection by phenylalanyl tRNA synthetase of tRNAPhe but not of Phe‐tRNAPhe; ATP abolished the shielding of the G‐C‐C‐A terminus of tRNASer by seryl tRNA synthetase.A number of tRNAPhe and tRNASer fragments was studied. It was concluded that the terminal 7 nucleotides in tRNAPhe are not required for complex formation. As expected, combinations of homologous tRNA half molecules resembled the intact tRNAs in the degree of protection. When the half molecules were tested separately or in heterologous combinations, phenylalanyl tRNA synthetase afforded some protection for the pG‐half molecule of tRNAPhe and seryl tRNA synthetase for the C‐C‐A‐half molecule of tRNASer.