Mechanism of Discrimination between Cognate and Non-cognate tRNs by Phenylalanyl-tRNA:Synthetase from Yeast

Abstract

The interaction between phenylalanyl‐tRNA synthetase from yeast and Escherichia coli and tRNA^Phe (yeast), tRNA^Ser (yeast), tRNA^Val₁ (E. coli) and tRNA^Tyr (E. coli) has been investigated by ultracentrifugation analysis, fluorescence titration and fast kinetic techniques. The fluorescence of the Y‐base of tRNAP^Phe and the intrinsic fluorescence of the synthetases have been used as optical indicators. 1 Specific complexes between phenylalanyl‐tRNA synthetase and tRNA^Phe from yeast are formed in a two‐step mechanism: a nearly diffusion‐controlled recombination is followed by a fast conformational transition. Binding constants, rate constants and changes in the quantum yield of the Y‐base fluorescence upon binding are given under a variety of conditions with respect to pH, added salt, concentration of Mg²⁺ ions and temperature. 2 Heterologous complexes between phenylalany1‐tRNA synthetase (E. coli) and tRNA^Phe (yeast) are formed, in a similar two‐step mechanism as the specific complexes; the conformational transition, however, is slower by a factor 4–5. 3 Formation of non‐specific complexes between phenylalanyl‐tRNA synthetase (yeast) and tRNAT^Tyr (E. coli) proceeds in a one‐step mechanism. Phenylalanyl‐tRNA synthetase (yeast) binds either two molecults of tRNA^Phe (yeast) or only one molecule of tRNA^Tyr (E. coli); tRNA^Va1₁ (E. coli) or tRNA^Ser (yeast) are also bound in a 1:1 stoichiometry. Binding constants for complexes of phenylalanyl‐tRNA synthetase (yeast) and tRNA^Tyr (E. Coli) are determined under a variety of conditions. In contrast to specific complex formation, non‐specific binding is disfavoured by the presence of Mg²⁺ ions, and is not affected by pH and the presence of pyrophosphate. The difference in the stabilities of specific and non‐specific complexes can be varied by a factor of 2‐100 depending on the ionic conditions. Discrimination of cognate and non‐cognate tRNA by:‐phenylalanyl‐tRNA synthetase (yeast) is discussed in terms of the binding mechanism, the topology of the binding sites, the nature of interacting forces and the relation between specificity and ionic conditions.