Evidence for two types of complexes formed by yeast tyrosyl‐tRNA synthetase with cognate and non‐cognate tRNA

Polyacrylamide gel electrophoresis at pH 8.3 was used to detect and quantitate the formation of the yeast tyrosyl‐tRNA synthetase (an α 2 ‐type enzyme) complex with its cognate tRNA. Electrophoretic mobility of the complex is intermediate between the free enzyme and free tRNA; picomolar quantities can be readily detected by silver staining and quantitated by densitometry of autoradiograms when [ 32 P]tRNA is used. Two kinds of complexes of Tyr‐tRNA synthetase with yeast tRNA Tyr were detected. A slower‐moving complex is formed at ratios of tRNA Tyr /enzyme ≤ 0.5; it is assigned the composition tRNA · (α 2 ) 2 . At higher ratios, a faster‐moving complex is formed, approaching saturation at tRNA Tyr /enzyme = 1; any excess of tRNA Tyr remains unbound. This complex is assigned the composition tRNA ·α 2 . The slower, i.e. tRNA · (α 2 ) 2 complex, but not the faster complex, can be formed even with non‐cognate tRNAs. Competition experiments show that the affinity of the enzyme towards tRNA Tyr is at least 10‐fold higher than that for the non‐cognate tRNAs. ATP and GTP affect the electrophoretic mobility of the enzyme and prevent the formation of tRNA · (α 2 ) 2 complexes both with cognate and non‐cognate tRNAs, while neither tyrosine, as the third substrate of Tyr tRNA synthetase, nor AMP, AMP/PP i , or spermidine, have such effects. Hence, the ATP‐mediated formation of the α 2 structure parallels the increase in specificity of the enzyme towards its cognate tRNA.