Abstract
The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement.
Highlights
The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs
Molecular dynamics simulations suggest that fluctuations of most other ribosomal elements are rapid and spontaneous tRNA movement through the ribosome is restricted by the coupled dynamics of the tRNA2–mRNA module[18]
Little is known about the order of appearance of the various chimeric intermediates during unperturbed translocation, their dynamic properties, functional role, or how they contribute to the energy landscape of elongation factor G (EF-G)-catalysed translocation
Summary
The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. In the POST state, the peptidyl-tRNA occupies the P/P position and the ribosome assumes the classical, non-rotated conformation At this stage, the peptidyl-tRNA has gained the ability to rapidly react with puromycin (Pmn), a mimic of the 30-end of aminoacyl-tRNA used as a tool for monitoring 50S translocation. EF-G induces the formation of further sub-states, with tRNAs gradually moving with respect to the 30S and 50S subunits, designated, for example, ap/P, A/P2 or ap/ap states[23,30,31] These intermediates with tRNAs in so-called chimeric states can be isolated by stalling the ribosome–tRNA–EF-G complex using (i) antibiotics, which inhibit translocation at different stages and by different mechanisms[32]; (ii) non-hydrolysable GTP analogues, which trap. Little is known about the order of appearance of the various chimeric intermediates during unperturbed translocation, their dynamic properties, functional role, or how they contribute to the energy landscape of EF-G-catalysed translocation
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