Single-Molecule Fluorescence Measurements of Ribosomal Translocation Dynamics

SummaryWe employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G⋅GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA⋅EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.

Graphical AbstractFigure optionsDownload high-quality image (184 K)Download as PowerPoint slideHighlights
► Concerted L11-tRNA and tRNA-tRNA distance fluctuations: classical/hybrid PRE states
► EF-G binds directly to both classical and hybrid PRE states
► EF-G binding halts L11-tRNA and tRNA-tRNA distance fluctuations prior to translocation
► Translocation from the classical state proceeds via a short-lived hybrid intermediate