Translocation requires large-scale movements of ribosome-bound tRNAs. Using tRNAs that are proflavin labeled and single-turnover rapid kinetics assays, we identify one or possibly two kinetically competent intermediates in translocation. EF-G.GTP binding to the pretranslocation (PRE) complex and GTP hydrolysis are rapidly followed by formation of the securely identified intermediate complex (INT), which is more slowly converted to the posttranslocation (POST) complex. Peptidyl tRNA within the INT complex occupies a hybrid site, which has a puromycin reactivity intermediate between those of the PRE and POST complexes. Thiostrepton and viomycin inhibit INT formation, whereas spectinomycin selectively inhibits INT disappearance. The effects of other translocation modulators suggest that EF-G-dependent GTP hydrolysis is more important for INT complex formation than for INT complex conversion to POST complex and that subtle changes in tRNA structure influence coupling of tRNA movement to EF-G.GTP-induced conformational changes.

The 30 S subunit contains 2 sites for tRNA binding (Phe-tRNA, AcPhe-tRNA, tRNA%i with the functional properties of D and A sites of the 70 S ribosome after attachment of 50 S subunit. The third (E) site specific for deacylated tRNA is introduced into 70 S ribosome by its 50 S subunit. The E-site binding of tRNA& is not sensitive to either tetracycline and edeine, and practically codon-independent. The affinity constant of tRNA@ for the E site is 2-3 orders of magnitude lower than that for the D site.

The L shape of tRNA is stabilized by the 'tertiary core' region, which contains base-pairing interactions between the D and T loops. Distortions of the L shape accompany tRNA movement across the ribosomal surface. Here, using single-turnover rapid kinetics assays, we determine the effects of mutations within the tertiary core of P site-bound tRNA(fMet) on three measures of the rate of translocation, the part of the elongation cycle involving the most extensive tRNA movement. Mutations in the strictly conserved G18.U55 base pair result in as much as an 80-fold decrease in the rate of translocation, demonstrating the importance of the 18-55 interaction for rapid translocation. This implicates the core region as a locus for functionally important dynamic interactions with the ribosome and leads to the proposal that translocation of ribosome-bound tRNAs may be sequential rather than concerted.