title: Coordinated conformational and compositional dynamics drive ribosome translocation.
abstract: During translation elongation, the ribosome compositional factors elongation factor G (EF-G; encoded by fusA) and tRNA alternately bind to the ribosome to direct protein synthesis and regulate the conformation of the ribosome. Here, we use single-molecule fluorescence with zero-mode waveguides to directly correlate ribosome conformation and composition during multiple rounds of elongation at high factor concentrations in Escherichia coli. Our results show that EF-G bound to GTP (EF-G-GTP) continuously samples both rotational states of the ribosome, binding with higher affinity to the rotated state. Upon successful accommodation into the rotated ribosome, the EF-G-ribosome complex evolves through several rate-limiting conformational changes and the hydrolysis of GTP, which results in a transition back to the nonrotated state and in turn drives translocation and facilitates release of both EF-G-GDP and E-site tRNA. These experiments highlight the power of tracking single-molecule conformation and composition simultaneously in real time.
authors.affiliation: Department of Applied Physics, Stanford University, Stanford, California, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Chen
authors.forename: Alexey
authors.initials: A
authors.lastname: Petrov
authors.forename: Albert
authors.initials: A
authors.lastname: Tsai
authors.forename: Seán E
authors.initials: SE
authors.lastname: O'Leary
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Puglisi
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D008954:Models, Biological; D020653:Peptide Elongation Factor G / Q000378:metabolism*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012343:RNA, Transfer / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D020653:Peptide Elongation Factor G; D006160:Guanosine Triphosphate; D012343:RNA, Transfer