title: Ribosomal initiation complex-driven changes in the stability and dynamics of initiation factor 2 regulate the fidelity of translation initiation.
abstract: Joining of the large, 50S, ribosomal subunit to the small, 30S, ribosomal subunit initiation complex (IC) during bacterial translation initiation is catalyzed by the initiation factor (IF) IF2. Because the rate of subunit joining is coupled to the IF, transfer RNA (tRNA), and mRNA codon compositions of the 30S IC, the subunit joining reaction functions as a kinetic checkpoint that regulates the fidelity of translation initiation. Recent structural studies suggest that the conformational dynamics of the IF2·tRNA sub-complex forming on the intersubunit surface of the 30S IC may play a significant role in the mechanisms that couple the rate of subunit joining to the IF, tRNA, and codon compositions of the 30S IC. To test this hypothesis, we have developed a single-molecule fluorescence resonance energy transfer signal between IF2 and tRNA that has enabled us to monitor the conformational dynamics of the IF2·tRNA sub-complex across a series of 30S ICs. Our results demonstrate that 30S ICs undergoing rapid subunit joining display a high affinity for IF2 and an IF2·tRNA sub-complex that primarily samples a single conformation. In contrast, 30S ICs that undergo slower subunit joining exhibit a decreased affinity for IF2 and/or a change in the conformational dynamics of the IF2·tRNA sub-complex. These results strongly suggest that 30S IC-driven changes in the stability of IF2 and the conformational dynamics of the IF2·tRNA sub-complex regulate the efficiency and fidelity of subunit joining during translation initiation.
authors.affiliation: Department of Chemistry, Columbia University, 3000 Broadway, MC3126, New York, NY 10027, USA.
authors.forename: Jiangning
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Columbia University, 3000 Broadway, MC3126, New York, NY 10027, USA.
authors.forename: Kelvin
authors.initials: K
authors.lastname: Caban
authors.affiliation: Department of Chemistry, Columbia University, 3000 Broadway, MC3126, 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*; 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; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; D006160:Guanosine Triphosphate
keywords: 30S IC; binding kinetics; conformational dynamics; smFRET; translation initiation