title: Initiation factor 2 stabilizes the ribosome in a semirotated conformation.
abstract: Intersubunit rotation and movement of the L1 stalk, a mobile domain of the large ribosomal subunit, have been shown to accompany the elongation cycle of translation. The initiation phase of protein synthesis is crucial for translational control of gene expression; however, in contrast to elongation, little is known about the conformational rearrangements of the ribosome during initiation. Bacterial initiation factors (IFs) 1, 2, and 3 mediate the binding of initiator tRNA and mRNA to the small ribosomal subunit to form the initiation complex, which subsequently associates with the large subunit by a poorly understood mechanism. Here, we use single-molecule FRET to monitor intersubunit rotation and the inward/outward movement of the L1 stalk of the large ribosomal subunit during the subunit-joining step of translation initiation. We show that, on subunit association, the ribosome adopts a distinct conformation in which the ribosomal subunits are in a semirotated orientation and the L1 stalk is positioned in a half-closed state. The formation of the semirotated intermediate requires the presence of an aminoacylated initiator, fMet-tRNA(fMet), and IF2 in the GTP-bound state. GTP hydrolysis by IF2 induces opening of the L1 stalk and the transition to the nonrotated conformation of the ribosome. Our results suggest that positioning subunits in a semirotated orientation facilitates subunit association and support a model in which L1 stalk movement is coupled to intersubunit rotation and/or IF2 binding.
authors.affiliation: Department of Biochemistry and Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642.
authors.forename: Clarence
authors.initials: C
authors.lastname: Ling
authors.affiliation: Department of Biochemistry and Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642 Dmitri_Ermolenko@urmc.rochester.edu.
authors.forename: Dmitri N
authors.initials: DN
authors.lastname: Ermolenko
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006160:Guanosine Triphosphate / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008968:Molecular Conformation; D039861:Prokaryotic Initiation Factor-1 / Q000378:metabolism; D039881:Prokaryotic Initiation Factor-2 / Q000378:metabolism*; D039665:Prokaryotic Initiation Factor-3 / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000235:genetics / Q000378:metabolism; D054657:Ribosome Subunits / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D039861:Prokaryotic Initiation Factor-1; D039881:Prokaryotic Initiation Factor-2; D039665:Prokaryotic Initiation Factor-3; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; D006160:Guanosine Triphosphate
keywords: initiation factor 2; ribosome; single-molecule FRET; translation initiation