Single-Molecule Fluorescence Measurements of Ribosomal Translocation Dynamics

Abstract

We 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. Pre-translocation (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 post-translocation (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.

The conserved protein EF4 (LepA) modulates the elongation cycle of protein synthesis

“…As determined from both fluorescence change and puromycin reactivity measurements, an EF4/EF-G ratio of 0.1-0.2 suffices to achieve half conversion of PRE complex to X 3 , suggesting an apparent affinity of EF4 for PRE complex that is some 5-10 times higher than that of EF-G. Single molecule FRET (smFRET) studies have shown that PRE complexes are distributed between hybrid and classical states (2,3) and that a fraction of these complexes fluctuate between these states (2,22). Following Chen et al (22), we used smFRET to examine PRE complexes formed using two different sets of fluorescently labeled materials.…”

“…Single molecule FRET (smFRET) studies have shown that PRE complexes are distributed between hybrid and classical states (2,3) and that a fraction of these complexes fluctuate between these states (2,22). Following Chen et al (22), we used smFRET to examine PRE complexes formed using two different sets of fluorescently labeled materials. PRE-Lt complex contains L11 labeled with Cy3 at position 87 (L11 Cy3 ) and fMetPhe-tRNA Phe (Cy5) in the A site and unlabeled tRNA fMet in the P site.…”

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

“…Elongation factor 2 in complex with GTP binds to the PRE ribosome and, regardless of its initial conformation, causes an appearance of the hybrid tRNA states (5,10). After binding, the protein undergoes conformational changes that bring the second superdomain into the A-site of the SSU (11,12), where it interacts via domain IV with the decoding centre and tRNA-mRNA-complex (12)(13)(14).…”

Bacterial replication, transcription and translation: mechanistic insights from single-molecule biochemical studies

“…Attaching two differently coloured dyes to a system of interest also allows single-molecule Förster (fluorescence) resonance energy transfer (smFRET) to be measured 12 . This approach facilitates the real-time measurement of dye-to-dye distances of up to ~5 nm.…”