Aminoacylation kinetics and specificity for viral genomic RNAs

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Virginia Polytechnic Institute and State University

The esterification of amino acids to the tRNA-like structures of viral genomic RNA was studied as catalyzed by amino acyl tRNA synthetases from E. coli, yeast, bean and rat. Peanut stunt virus RNA was esterified with tyrosine in the presence of bean tyrosyl-tRNA synthetase to a maximum of 22 mole percent (average MW = 1 x 10⁶ ). The rate of TYMV-RNA aminoacylation was extensively studied. In the presence of enzymes from E. coli, yeast and bean, the rate of aminoacylation was inhibited 4 fold by 47.5 mM KCl. In the presence of rat enzyme a 1.5 fold increase in rate was observed. In identical studies using tRNA, added KCl generally favored aminoacylation of tRNA by the homologous enzyme but disfavored heterologous reactions.

The kinetic parameters (Km and Vmax of TYMV-RNA aminoacylation in the presence of valyl-tRNA synthetases from the four sources were determined and compared to results obtained for yeast and E. coli tRNA controls. TYMV-RNA was found to be a uniquely competent and versatile substrate as compared to tRNA. The Km of yeast valyl-tRNA synthetase for TYMV-RNA (7 nM) is the second lowest Km reported for any aminoacyl tRNA synthetase. Kinetic studies using TYMV-RNA with or without the 3' terminal AMP indicate that this AMP is involved in the binding of TYMV-RNA to yeast valyl-tRNA synthetase.