title: 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. 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.
authors.affiliation: Pennsylvania Muscle Institute, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Chunlai
authors.initials: C
authors.lastname: Chen
authors.forename: Benjamin
authors.initials: B
authors.lastname: Stevens
authors.forename: Jaskarin
authors.initials: J
authors.lastname: Kaur
authors.forename: Diana
authors.initials: D
authors.lastname: Cabral
authors.forename: Hanqing
authors.initials: H
authors.lastname: Liu
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Wang
authors.forename: Haibo
authors.initials: H
authors.lastname: Zhang
authors.forename: Gabriel
authors.initials: G
authors.lastname: Rosenblum
authors.forename: Zeev
authors.initials: Z
authors.lastname: Smilansky
authors.forename: Yale E
authors.initials: YE
authors.lastname: Goldman
authors.forename: Barry S
authors.initials: BS
authors.lastname: Cooperman
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002232:Carbocyanines / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007700:Kinetics; D008956:Models, Chemical; D008957:Models, Genetic; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D020653:Peptide Elongation Factor G / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002232:Carbocyanines; D020653:Peptide Elongation Factor G; D012269:Ribosomal Proteins; C089187:cyanine dye 3; C085321:cyanine dye 5; C027624:ribosomal protein L11; D000255:Adenosine Triphosphate; D012343:RNA, Transfer