title: Spontaneous formation of the unlocked state of the ribosome is a multistep process.
abstract: The mechanism of substrate translocation through the ribosome is central to the rapid and faithful translation of mRNA into proteins. The rate-limiting step in translocation is an unlocking process that includes the formation of an "unlocked" intermediate state, which requires the convergence of large-scale conformational events within the ribosome including tRNA hybrid states formation, closure of the ribosomal L1 stalk domain, and subunit ratcheting. Here, by imaging of the pretranslocation ribosome complex from multiple structural perspectives using two- and three-color single-molecule fluorescence resonance energy transfer, we observe that tRNA hybrid states formation and L1 stalk closure, events central to the unlocking mechanism, are not tightly coupled. These findings reveal that the unlocked state is achieved through a stochastic-multistep process, where the extent of conformational coupling depends on the nature of tRNA substrates. These data suggest that cellular mechanisms affecting the coupling of conformational processes on the ribosome may regulate the process of translation elongation.
authors.affiliation: Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, NY, USA.
authors.forename: James B
authors.initials: JB
authors.lastname: Munro
authors.forename: Roger B
authors.initials: RB
authors.lastname: Altman
authors.forename: Chang-Shung
authors.initials: CS
authors.lastname: Tung
authors.forename: Jamie H D
authors.initials: JH
authors.lastname: Cate
authors.forename: Kevin Y
authors.initials: KY
authors.lastname: Sanbonmatsu
authors.forename: Scott C
authors.initials: SC
authors.lastname: Blanchard
mesh_terms: D017124:Conserved Sequence; D031541:Fluorescence Resonance Energy Transfer; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics; D012358:RNA, Transfer, Met / Q000737:chemistry; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D011506:Proteins; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; D012343:RNA, Transfer; D020558:GTP Phosphohydrolases