title: Translation initiation factor 3 regulates switching between different modes of ribosomal subunit joining.
abstract: Ribosomal subunit joining is a key checkpoint in the bacterial translation initiation pathway during which initiation factors (IFs) regulate association of the 30S initiation complex (IC) with the 50S subunit to control formation of a 70S IC that can enter into the elongation stage of protein synthesis. The GTP-bound form of IF2 accelerates subunit joining, whereas IF3 antagonizes subunit joining and plays a prominent role in maintaining translation initiation fidelity. The molecular mechanisms through which IF2 and IF3 collaborate to regulate the efficiency of 70S IC formation, including how they affect the dynamics of subunit joining, remain poorly defined. Here, we use single-molecule fluorescence resonance energy transfer to monitor the interactions between IF2 and the GTPase-associated center (GAC) of the 50S subunit during real-time subunit joining reactions in the absence and presence of IF3. In the presence of IF3, IF2-mediated subunit joining becomes reversible, and subunit joining events cluster into two distinct classes corresponding to formation of shorter- and longer-lifetime 70S ICs. Inclusion of IF3 within the 30S IC was also found to alter the conformation of IF2 relative to the GAC, suggesting that IF3's regulatory effects may stem in part from allosteric modulation of IF2-GAC interactions. The results are consistent with a model in which IF3 can exert control over the efficiency of subunit joining by modulating the conformation of the 30S IC, which in turn influences the formation of stabilizing intersubunit contacts and thus the reaction's degree of reversibility.
authors.affiliation: Columbia University Department of Chemistry, 3000 Broadway, New York, NY 10027, USA.
authors.forename: Daniel D
authors.initials: DD
authors.lastname: MacDougall
authors.affiliation: Columbia University Department of Chemistry, 3000 Broadway, New York, NY 10027, USA. Electronic address: rlg2118@columbia.edu.
authors.forename: Ruben L
authors.initials: RL
authors.lastname: Gonzalez
mesh_terms: D004926:Escherichia coli / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D039881:Prokaryotic Initiation Factor-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D039665:Prokaryotic Initiation Factor-3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D012358:RNA, Transfer, Met / Q000378:metabolism; D054680:Ribosome Subunits, Small, Bacterial / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D039881:Prokaryotic Initiation Factor-2; D039665:Prokaryotic Initiation Factor-3; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; D006160:Guanosine Triphosphate
keywords: 70S initiation complex; GTPase-associated center; ribosome; smFRET; translation initiation