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

Open Access
Publisher Website
Google Scholar
Abstract
The interaction between phenylalanyl‐tRNA synthetase from yeast and Escherichia coli and tRNAPhe (yeast), tRNASer (yeast), tRNAVal1 (E. coli) and tRNATyr (E. coli) has been investigated by ultracentrifugation analysis, fluorescence titration and fast kinetic techniques. The fluorescence of the Y‐base of tRNAPPhe and the intrinsic fluorescence of the synthetases have been used as optical indicators. 1 Specific complexes between phenylalanyl‐tRNA synthetase and tRNAPhe 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 tRNAPhe (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 tRNATTyr (E. coli) proceeds in a one‐step mechanism. Phenylalanyl‐tRNA synthetase (yeast) binds either two molecults of tRNAPhe (yeast) or only one molecule of tRNATyr (E. coli); tRNAVa11 (E. coli) or tRNASer (yeast) are also bound in a 1:1 stoichiometry. Binding constants for complexes of phenylalanyl‐tRNA synthetase (yeast) and tRNATyr (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.