Late events of translation initiation in bacteria: a kinetic analysis.
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 P(i) release from the complex (1.5/s). The latter step was rate limiting for subsequent A-site binding of EF-Tu small middle dotGTP small middle dotPhe-tRNA(Phe) 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-tRNA(fMet) in the P site nor the release of IF2 from the ribosome required GTP hydrolysis.
Tomsic
J
J
Laboratory of Genetics, Department of Biology MCA, University of Camerino, 62032 Camerino (MC), Italy.
Vitali
L A
LA
Daviter
T
T
Savelsbergh
A
A
Spurio
R
R
Striebeck
P
P
Wintermeyer
W
W
Rodnina
M V
MV
Gualerzi
C O
CO
D003062:Codon / Q000235:genetics; D004151:Dipeptides / Q000096:biosynthesis / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005453:Fluorescence; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D009239:N-Formylmethionine / Q000378:metabolism; D010441:Peptide Chain Elongation, Translational; D010442:Peptide Chain Initiation, Translational; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D039881:Prokaryotic Initiation Factor-2; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000235:genetics*; D012358:RNA, Transfer, Met / Q000235:genetics / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003062:Codon; D004151:Dipeptides; D010448:Peptide Initiation Factors; D010710:Phosphates; D039881:Prokaryotic Initiation Factor-2; D012358:RNA, Transfer, Met; D012360:RNA, Transfer, Phe; C089238:fMet-tRNA(fMet); D006153:Guanosine Diphosphate; D009239:N-Formylmethionine; D010649:Phenylalanine; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu