The region around position 1067 in domain II of 23S rRNA frequently is referred to as the GTPase center of the ribosome. The notion is based on the observation that the binding of the antibiotic thiostrepton to this region inhibited GTP hydrolysis by elongation factor G (EF-G) on the ribosome at the conditions of multiple turnover. In the present work, we have reanalyzed the mechanism of action of thiostrepton. Results obtained by biochemical and fast kinetic techniques show that thiostrepton binding to the ribosome does not interfere with factor binding or with single-round GTP hydrolysis. Rather, the antibiotic inhibits the function of EF-G in subsequent steps, including release of inorganic phosphate from EF-G after GTP hydrolysis, tRNA translocation, and the dissociation of the factor from the ribosome, thereby inhibiting the turnover reaction. Structurally, thiostrepton interferes with EF-G footprints in the ␣-sarcin stem loop (A2660, A2662) located in domain VI of 23S rRNA. The results indicate that thiostrepton inhibits a structural transition of the 1067 region of 23S rRNA that is important for functions of EF-G after GTP hydrolysis.

The anticodon loop of tRNA contains a number of conserved or semiconserved nucleotides. In most tRNAs, a highly modified purine is found at position 37 immediately 3 to the anticodon. Here, we examined the role of the base at position 37 for tRNA Phe

During the translocation step of protein synthesis, a complex of two transfer RNAs bound to messenger RNA (tRNA-mRNA) moves through the ribosome. The reaction is promoted by an elongation factor, called EF-G in bacteria, which, powered by GTP hydrolysis, induces an open, unlocked conformation of the ribosome that allows for spontaneous tRNA-mRNA movement. Here we show that, in the absence of EF-G, there is spontaneous backward movement, or retrotranslocation, of two tRNAs bound to mRNA. Retrotranslocation is driven by the gain in affinity when a cognate E-site tRNA moves into the P site, which compensates the affinity loss accompanying the movement of peptidyl-tRNA from the P to the A site. These results lend support to the diffusion model of tRNA movement during translocation. In the cell, tRNA movement is biased in the forward direction by EF-G, which acts as a Brownian ratchet and prevents backward movement.

For the functional role of the ribosomal tRNA exit (E) site, two different models have been proposed. It has bleen suggested that transient E-site binding ofthe tRNA leaving the peptidyl (P) site promotes elongation factor G (EF-G)-dependent translocation by lowering the energetic barrier of tRNA release [Lill, R., Robertson, J. M. & Wintermeyer, W. (1989) EMBO J. 8,[3933][3934][3935][3936][3937][3938]. The alternative "allosteric three-site model" [Nierhaus, K. H. (1990) Textbook models of protein elongation distinguish two main states of the elongating ribosome: (i) the posttranslocation state with peptidyl-tRNA in the peptidyl (P) site and an empty aminoacyl (A) site, and (ii) the pretranslocation state with deacylated tRNA in the P site and peptidyl-tRNA in the A site. The latter state is formed by peptidyl transfer from the P site-bound peptidyl-tRNA to the aminoacyl-tRNA brought to the A site by the action of elongation factor Tu (EF-Tu). Translocation, that is the displacement of peptidyl-tRNA from the A site to the P site and the release of deacylated tRNA from the P site into solution, is brought about by elongation factor G (EF-G) that, during the reaction, hydrolyzes GTP (1, 2).For the molecular mechanism of tRNA translocation, a number of transient intermediate states have to be considered. Most significantly, a third tRNA binding site, the exit (E) site, has been demonstrated on Escherichia coli ribosomes that is implicated in the release of the deacylated tRNA from the P site during translocation (3-6). The E site has been described also for ribosomes from an archaeon (7), from rabbit (8, 9), and from yeast (10). The E site of E. coli ribosomes specifically binds deacylated tRNA with intermediate affinity (Kd = 0.1 to 50 ,uM, depending on conditions) in a kinetically labile fashion. The binding to a large extent depends on the interaction of the 3'-terminal adenosine with the ribosome (4, 11), presumably with 23S rRNA. In comparison, the contribution of cognate codon-anticodon interaction to the free energy of tRNA binding to the E site is small (12-14) and difficult to assess, since the intrinsic affinities of various tRNAs for binding to the E site vary by about the same order of magnitude, about 10-fold (13).For the functional role of the E site, two fundamentally different models have been put forward. We have provided evidence suggesting that transient E-site binding promotes the exit of the deacylated tRNA from the P site during translocation (13,15,16). The E site-bound intermediate state of the tRNA develops from the "hybrid" P/E state (17) by the action of EF-G and initiates translocation (18). In this model, the E site-bound state of the leaving tRNA, due to its low kinetic stability, is considered a transient intermediate rather than a stable product of translocation, and, therefore, a feature of the molecular mechanism of translocation.In contrast, Nierhaus and colleagues (review ref. 19) have proposed that, after translocation, the deacylated tRNA remains stably bound to the...