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

Complexes of tRNA Phe and tRNA Ser 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. tRNA Phe was found to be strongly protected by yeast phenylalanyl tRNA synthetase at both nuclease‐sensitive sites, the dihydrouridine loop and the anticodon. tRNA Ser , 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 tRNA Phe , seryl tRNA synthetase two moles of tRNA Ser against nuclease attack. Denatured tRNA Phe 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 tRNA Phe but not of Phe‐tRNA Phe ; ATP abolished the shielding of the G‐C‐C‐A terminus of tRNA Ser by seryl tRNA synthetase. A number of tRNA Phe and tRNA Ser fragments was studied. It was concluded that the terminal 7 nucleotides in tRNA Phe 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 tRNA Phe and seryl tRNA synthetase for the C‐C‐A‐half molecule of tRNA Ser .