title: Activation of initiation factor 2 by ligands and mutations for rapid docking of ribosomal subunits.
abstract: We previously identified mutations in the GTPase initiation factor 2 (IF2), located outside its tRNA-binding domain, compensating strongly (A-type) or weakly (B-type) for initiator tRNA formylation deficiency. We show here that rapid docking of 30S with 50S subunits in initiation of translation depends on switching 30S subunit-bound IF2 from its inactive to active form. Activation of wild-type IF2 requires GTP and formylated initiator tRNA (fMet-tRNA(i)). In contrast, extensive activation of A-type IF2 occurs with only GTP or with GDP and fMet-tRNA(i), implying a passive role for initiator tRNA as activator of IF2 in subunit docking. The theory of conditional switching of GTPases quantitatively accounts for all our experimental data. We find that GTP, GDP, fMet-tRNA(i) and A-type mutations multiplicatively increase the equilibrium ratio, K, between active and inactive forms of IF2 from a value of 4 × 10(-4) for wild-type apo-IF2 by factors of 300, 8, 80 and 20, respectively. Functional characterization of the A-type mutations provides keys to structural interpretation of conditional switching of IF2 and other multidomain GTPases.
authors.affiliation: Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, Sweden.
authors.forename: Michael Y
authors.initials: MY
authors.lastname: Pavlov
authors.forename: Anna
authors.initials: A
authors.lastname: Zorzet
authors.forename: Dan I
authors.initials: DI
authors.lastname: Andersson
authors.forename: Måns
authors.initials: M
authors.lastname: Ehrenberg
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli; D006160:Guanosine Triphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D039881:Prokaryotic Initiation Factor-2 / Q000235:genetics* / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000235:genetics*; D012358:RNA, Transfer, Met / Q000378:metabolism; D054657:Ribosome Subunits / Q000378:metabolism*; D012486:Salmonella typhimurium; D017422:Sequence Analysis, DNA; D013045:Species Specificity
chemical_list: D039881:Prokaryotic Initiation Factor-2; D012358:RNA, Transfer, Met; C089238:fMet-tRNA(fMet); D006160:Guanosine Triphosphate