GTPase center of elongation factor Tu is activated by occupation of the second tRNA binding site

Interaction of the elongation factor EF-Tu with the antibiotic kirromycin results in activation of the GTPase center of the factor and in induction of an additional tRNA binding site (tRNA binding site II to distinguish it from the classical tRNA binding site I). Activation of the GTPase center under these conditions is stimulated by addition of tRNA. Two-fold evidence is presented that this stimulation is due to tRNA binding to site II rather than to site I. First, a strong correlation is observed between stimulation of the GTPase activity and enhancement of the reactivity of Cys-81 of EF-Tu toward N-ethylmaleimide at various concentrations of aminoacyl-tRNA, deacylated tRNA, and N-acetylaminoacyl-tRNA. The latter effects signal tRNA binding to site II. Stimulation of the kirromycin-induced GTPase activity by tRNA binding to the factor also occurs when binding to site I is completely abolished. Such an abolishment was achieved by treating EF-Tu extensively with the thiol reagent L-1-tosylamido-2-phenylethyl chloromethyl ketone. EF-Tu X GTP thus treated has lost its ability to protect the ester bond of aminoacyl-tRNA. The relevance of these data for the sequence of events during protein synthesis and for control of translational fidelity is discussed.