Recognition of the universally conserved 3′-CCA end of tRNA by elongation factor EF-Tu

Abstract

Escherichia coli tRNA^Val with pyrimidine substitutions for the universally conserved 3′-terminal adenine can be readily aminoacylated. It cannot, however, transfer valine into polypeptides. Conversely, despite being a poor substrate for valyl-tRNA synthetase, tRNA^Val with a 3′-terminal guanine is active in in vitro polypeptide synthesis. To better understand the function of the 3′-CCA sequence of tRNA in protein synthesis, the effects of systematically varying all three bases on formation of the Val-tRNA^Val:EF-Tu:GTP ternary complex were investigated. Substitutions at C74 and C75 have no significant effect, but replacing A76 with pyrimidines decreases the affinity of valyl-tRNA^Val for EF-Tu:GTP, thus explaining the inability of these tRNA^Val variants to function in polypeptide synthesis. Valyl-tRNA^Val terminating in 3′-guanine is readily recognized by EF-TU:GTP. Dissociation constants of the EF-Tu:GTP ternary complexes with valine tRNAs having nucleotide substitutions at the 3′ end increase in the order adenine < guanine < uracil; EF-Tu has very little affinity for tRNA terminating in 3′ cytosine. Similar observations were made in studies of the interaction of 3′ end mutants of E. coli tRNA^Ala and tRNA^Phe with EF-Tu:GTP. These results indicate that EF-Tu:GTP preferentially recognizes purines and discriminates against pyrimidines, especially cytosine, at the 3′ end of aminoacyl-tRNAs.