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 aminoacyl-tRNA. 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.

Figure 1

Schematic drawing of relationship between pre- and post-translocation state ribosomes and of known rRNA–tRNA pairing interactions. (a) Model of tRNA sampling between hybrid and classic state of tRNA binding before EF-G– or sparsomycin-mediated translocation. (b) Scheme of Watson-Crick base-pairing interactions between the P and A loops of the 23S rRNA and the CCA ends of tRNAs bound in the P and A site.

Figure 2

Toeprinting analysis of sparsomycin- and EF-G–mediated translocation on m301 messenger RNA. (a–d) Pretranslocation complexes were assembled by binding deacylated tRNA^Tyr, tRNA^Tyr C74G or tRNA^Tyr C75G to the P site (P lanes), then binding AcPhe-tRNA^Phe, AcPhe-tRNA^Phe C74G or AcPhe-tRNA^Phe C75G to the A site (A lanes). Complexes were incubated with sparsomycin (+Sps), 2.5% (v/v) DMSO (−Sps) or EF-G–GTP (+EF-G). Ribosomes used were either WT (a), G2252C (b), G2251C (c) or G2553C (d). Asterisk in a indicates the alternative, preferred binding site for AcPhe-tRNA^Phe on ribosomes programmed with m301 mRNA.

Figure 3

Analysis of sparsomycin-mediated translocation and puromycin reactivity of pretranslocation ribosome complexes carrying initiator and elongator tRNA^Met. (a) Sparsomycin-and EF-G–mediated translocation on MRE600 ribosomes and gene32 mRNA, monitored by toeprinting analysis. Pretranslocation complexes were assembled by binding deacylated tRNA^fMet, tRNA^Met, tRNA^fMet C74U or tRNA^fMet containing the C1G-A72C base pair (bp) to the P site (P lanes), then binding AcPhe-tRNA^Phe to the A site (A lanes). Complexes were incubated with sparsomycin (+Sps) or with 2.5% DMSO as a control (−Sps). (b) Hybrid reactivity of [¹⁴C]AcPhe-tRNA^Phe with puromycin, analyzed by electrophoretic TLC. Pretranslocation complexes prepared with deacylated initiator tRNA^fMet (lanes 1 and 2) or elongator tRNA^Met (lanes 3 and 4) in the P site and [¹⁴C]AcPhe-tRNA^Phe in the A site were incubated with (+) or without (−) puromycin. (c) Time course of reactivity of [¹⁴C]AcPhe-tRNA^Phe with puromycin in a pretranslocation complex with elongator tRNA^Met.

Figure 4

Pre–steady-state kinetic analysis of EF-G–mediated translocation. (a–d) Pretranslocation complexes with tRNA^fMet in the P site and dipeptidyl-tRNA in the A site were rapidly mixed with EF-G–GTP in the stopped-flow apparatus, and fluorescence was measured as a function of time. The A-site tRNAs used are indicated next to the trace (WT, C75G or C74G). The ribosomes used are indicated at top of panel (WT in a and b, G2252 in c and G2251 in d).