On the Specificity of Interactions between Transfer Ribonucleic Acids and Aminoacyl‐tRNA Synthetases

Abstract

The interactions of yeast seryl‐tRNA and phenylalanyl‐tRNA synthetases with the cognate and the noncognate tRNA have been studied by fluorescence spectroscopy. The binding process was followed by observing the changes in tryptophan fluorescence of the synthetases as well as the changes in the polarized fluorescence of the tRNAs covalently labeled with ethidium bromide, proflavine, or 1, N6‐etheno‐ATP. The degree of polarization of the fluorescence labels is particularly sensitive to changes in rotational diffusion of the tRNA. Both methods can be regarded as complementary and gave comparable results. There exist, however, cases such as the interaction between seryl‐tRNA synthetase and tRNASer in the absence of Mg2+, where the binding process could only be detected by measurement of the fluorescence polarization.Unspecific interactions between synthetases and tRNAs (or tRNA‐dye compounds) could be detected and were found to be highly dependent on pH, salt concentration and on the buffer used. In potassium phosphate buffer pH 7.3 discrimination between cognate and noncognate tRNA by seryl‐tRNA synthetase increases with addition of salt. The tryptophan fluorescence of phenylalanyl‐tRNA synthetase was influenced differently by the cognate and the noncognate tRNA while the stabilities of the complexes between this synthetase and the ethidium‐labeled tRNAPhe and tRNASer were rather similar. Seryl‐tRNA synthetase apparently discriminates more efficiently than phenylalanyl‐tRNA synthetase between the cognate and the noncognate tRNAs.The presence of two binding sites on seryl‐tRNA synthetase and the presence of one binding site on phenylalanyl‐tRNA synthetase for cognate as well as noncognate tRNA could be deduced. For the specific interaction between tRNASer and seryl‐tRNA synthetase in the presence of Mg2+ indications for cooperative behavior have been found. Experiments on competition between noncognate and cognate tRNAs as well as tRNA half molecules for the binding to the synthetase indicate an overlap of specific and unspecific binding sites.We conclude that the unspecific interaction may be an important initial step preceding the specific binding and recognition of the tRNA by the synthetase.