Codon-dependent conformational change of elongation factor Tu preceding GTP hydrolysis on the ribosome.

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Abstract
The mechanisms by which elongation factor Tu (EF‐Tu) promotes the binding of aminoacyl‐tRNA to the A site of the ribosome and, in particular, how GTP hydrolysis by EF‐Tu is triggered on the ribosome, are not understood. We report steady‐state and time‐resolved fluorescence measurements, performed in the Escherichia coli system, in which the interaction of the complex EF‐Tu.GTP.Phe‐tRNAPhe with the ribosomal A site is monitored by the fluorescence changes of either mant‐dGTP [3′‐O‐(N‐methylanthraniloyl)‐2‐deoxyguanosine triphosphate], replacing GTP in the complex, or of wybutine in the anticodon loop of the tRNA. Additionally, GTP hydrolysis is measured by the quench‐flow technique. We find that codon‐anticodon interaction induces a rapid rearrangement within the G domain of EF‐Tu around the bound nucleotide, which is followed by GTP hydrolysis at an approximately 1.5‐fold lower rate. In the presence of kirromycin, the activated conformation of EF‐Tu appears to be frozen. The steps following GTP hydrolysis‐‐the switch of EF‐Tu to the GDP‐bound conformation, the release of aminoacyl‐tRNA from EF‐Tu to the A site, and the dissociation of EF‐Tu‐GDP from the ribosome‐‐which are altogether suppressed by kirromycin, are not distinguished kinetically. The results suggest that codon recognition by the ternary complex on the ribosome initiates a series of structural rearrangements resulting in a conformational change of EF‐Tu, possibly involving the effector region, which, in turn, triggers GTP hydrolysis.