On dealing with anomalies in the transfer RNA aminoacylation reaction in partially purified systems

Abstract

Apparent differences in tRNA and aminoacyl-tRNA synthetase complements in tissues undergoing differentiation have frequently been used to support theories of translational control. The validity of at least some of these studies must now be questioned because of anomalies in the tRNA aminoacylation reaction which can lead to incomplete aminoacylation of tRNA. Incomplete acylation of a tRNA mixture could result in different relative amounts of acylated isoaccepting species if acylation rates were not identical for all species. Using common methods of analysis, this situation could lead to misestimation of relative levels of isoacceptors or an inability to detect the presence of minor species. Bonnet and Ebel [Bonnet, J., and Ebel, J. (1972) Eur. J. Biochem. 31, 335] used a highly purified valyl-tRNA and valyl-tRNA synthetase from yeast to demonstrate the presence of four reactions that occur simultaneously in that system. Herein, I apply the findings of Bonnet and Ebel to a mammalian system in a manner which is representative of attempts to study relative tissue proportions of tRNA isoacceptors. Total complements of tRNAs and the aminoacyl-tRNA synthetases have been partially purified from rabbit liver according to the methods of Yang and Novelli [Yang, W. K., and Novelli, G. D. (1971) in Methods in Enzymology (Moldave, K., and Grossman, L., eds.), Vol. 20, p. 44, Academic Press, New York], probably the most commonly used procedures in the literature. Reaction conditions for tRNA acylation are shown to be modifiable so as to influence the extent of tRNA acylation. Procedures for optimizing the extent of tRNA acylation in such systems are demonstrated, and the unfavorable influence of Tris buffer, a factor not discussed by Bonnet and Ebel, is shown. Finally, examples of altered ratios of isoaccepting species in samples incompletely acylated due to suboptimal reaction conditions are provided.