The hybrid state of tRNA binding is an authentic translation elongation intermediate

Abstract

The GTPase elongation factor (EF)-G is responsible for promoting the translocation of the messenger RNA-transfer RNA complex on the ribosome, thus opening up the A site for the next aminoacyltRNA. Chemical modification and cryo-EM studies have indicated that tRNAs can bind the ribosome in an alternative 'hybrid' state after peptidyl transfer and before translocation, though the relevance of this state during translation elongation has been a subject of debate. Here, using pre-steady-state kinetic approaches and mutant analysis, we show that translocation by EF-G is most efficient when tRNAs are bound in a hybrid state, supporting the argument that this state is an authentic intermediate during translation.Translation elongation is the multistep process performed by the ribosome to sequentially add mRNA-encoded amino acids to the growing polypeptide chain. There are three iterated steps performed by the ribosome during the elongation cycle: (i) a tRNA-selection step in which the ribosome and elongation factor (EF)-Tu select the next aminoacyl-tRNA to enter the cycle; (ii) peptide-bond formation catalyzed in the active site of the large ribosomal subunit; and (iii) translocation of the mRNA-tRNA complex through the subunit interface region, facilitated by EF-G, with associated GTP hydrolysis. The tRNA substrates are at the heart of each of these steps, where they have key functional roles 1-3 .Early studies on the ribosome identified binding sites for two tRNA substrates, the A site for the aminoacyl-tRNA and the P site for the peptidyl-tRNA. Further biochemistry eventually identified a third site, the E or exit site, where deacylated tRNAs bind after loss of the peptidyl moiety and before release into solution 4 . An understanding of the order and timing with which tRNA substrates occupy these three sites (on both the large and small subunits) during the elongation cycle is central to a detailed molecular view of the process of translation.The hybrid state of tRNA binding on the ribosome was first proposed in the 1960s as an elegant way to understand how controlled tRNA movement through the ribosome might be accomplished 5 . The basic feature of such a hybrid state of binding is that tRNAs would move independently with respect to the two subunits of the ribosome during the steps of the translation cycle. Fluorescence studies provided early biochemical clues that such a state of tRNA binding might be populated during translation 6 , and subsequent chemical-modification analysis provided clear and detailed experimental support for the hybrid state 7 . These studies Correspondence should be addressed to R.G. (ragreen@jhmi.edu).. Note: Supplementary information is available on the Nature Structural & Molecular Biology website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.Published online at http://www.nature.com/nsmb/ Reprints and permissions information is available online at http://www.nature.com/reprints/index.html NIH Public Access Fig. 1a). Thus, 'hybr...

Role of hybrid tRNA-binding states in ribosomal translocation

“…12) and is the rate-determining step for the process. Steps 4 and 5 may be conformationally linked: thus, EF-G-dependent translocation is accelerated by either mutation of the 50S A site or lengthening of the peptidyl group (17), and each of these changes promotes the A/A-to-A/P transition (4).…”

“…Effects of antibiotics and ribosomal mutations indicated that, although both events are limited by the unlocking step, codon-anticodon movement and P i release are independent of each other and probably occur in random order (12)(13)(14). Finally, ribosomal rearrangements must occur to ''relock'' the tRNAs in their new sites, followed by release of EF-G⅐GDP from the POST complex, although the kinetics of these events have yet to be fully characterized.A number of studies suggest an important role for hybrid-state formation in the mechanism of EF-G-dependent translocation (11,(15)(16)(17). However, it remains unclear how the P/P-to-P/E and A/A-to-A/P transitions relate to the kinetically defined events of translocation.…”

“…A number of studies suggest an important role for hybrid-state formation in the mechanism of EF-G-dependent translocation (11,(15)(16)(17). However, it remains unclear how the P/P-to-P/E and A/A-to-A/P transitions relate to the kinetically defined events of translocation.…”

Insights into the molecular determinants of EF-G catalyzed translocation

“…1; Samaha et al 1995;Kim and Green 1999). While the specific interactions of tRNAs in classical positions are critical for both peptide bond formation (Lieberman and Dahlberg 1994;Weinger et al 2004;Brunelle et al 2006;Dorner et al 2006) as well as peptide release (Feinberg and Joseph 2006), such contacts must be extensively remodeled for tRNA substrates to move directionally through the ribosome during active translation (Agirrezabala and Frank 2009;Munro et al 2009). …”

Molecular mechanics of 30S subunit head rotation

“…Translocation can be broadly divided into two steps. First, the tRNAs move on the large subunit from their classical A/A and P/P states to their hybrid A/P and P/E states (1,2), facilitated by intersubunit rotation, which can occur spontaneously and reversibly (3)(4)(5)(6)(7). During the second step, which is rate limiting and EF-G-dependent (8,9), the tRNA anticodon stem-loops (ASLs) and their associated mRNAs move from the A to P and P to E sites of the small subunit, thereby advancing the tRNAs into their classical P/P and E/E states.…”