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| The EMBO Journal
(2000) 19, 2127–2136, doi:10.1093/emboj/19.9.2127 |
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| Late events of translation initiation in bacteria: a kinetic analysis |
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Jerneja Tom ic1, Luca A. Vitali1, Tina Daviter2, Andreas Savelsbergh2, Roberto Spurio1, Petra Striebeck2, Wolfgang Wintermeyer2, Marina V. Rodnina2 and Claudio O. Gualerzi1
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1 Laboratory of Genetics, Department of Biology MCA, University of Camerino, 62032 Camerino (MC), Italy 2 Institute of Molecular Biology, University Witten/Herdecke, D-58448 Witten, Germany To whom correspondence should be addressed Claudio O. Gualerzi, gualerzi@cambio.unicam.it Received 11 January 2000; Revised 3 March 2000; Accepted 7 March 2000. |
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| Abstract |
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Binding of the 50S ribosomal subunit to the 30S initiation complex and the subsequent transition from the initiation to the elongation phase up to the synthesis of the first peptide bond represent crucial steps in the translation pathway. The reactions that characterize these transitions were analyzed by quench-flow and fluorescence stopped-flow kinetic techniques. IF2-dependent GTP hydrolysis was fast (30/s) followed by slow Pi release from the complex (1.5/s). The latter step was rate limiting for subsequent A-site binding of EF-Tu GTPPhe-tRNAPhe ternary complex. Most of the elemental rate constants of A-site binding were similar to those measured on poly(U), with the notable exception of the formation of the first peptide bond which occurred at a rate of 0.2/s. Omission of GTP or its replacement with GDP had no effect, indicating that neither the adjustment of fMet-tRNAfMet in the P site nor the release of IF2 from the ribosome required GTP hydrolysis. |
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| Keywords: A-site binding, fast kinetics, IF2-dependent GTPase, phosphate release, translation initiation |
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