Open AccessMechanism of Discrimination between Cognate and Non‐cognate tRNs by Phenylalanyl‐tRNA:Synthetase from Yeast
Author(s) -
KRAUSS Gerhard,
RIESNER Detlev,
MAASS Guenter
Publication year - 1976
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1976.tb10766.x
Subject(s) - transfer rna , yeast , protein quaternary structure , escherichia coli , binding site , biochemistry , chemistry , saccharomyces cerevisiae , biology , stereochemistry , rna , protein subunit , gene
The interaction between phenylalanyl‐tRNA synthetase from yeast and Escherichia coli and tRNA Phe (yeast), tRNA Ser (yeast), tRNA Val 1 (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 Mg 2+ 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 Va1 1 (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 Mg 2+ 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.