Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome.
The kinetic mechanism of elongation factor Tu (EF-Tu)-dependent binding of Phe-tRNAPhe to the A site of poly(U)-programmed Escherichia coli ribosomes has been established by pre-steady-state kinetic experiments. Six steps were distinguished kinetically, and their elemental rate constants were determined either by global fitting, or directly by dissociation experiments. Initial binding to the ribosome of the ternary complex EF-Tu.GTP.Phe-tRNAPhe is rapid (k1 = 110 and 60/micromM/s at 10 and 5 mM Mg2+, 20 degreesC) and readily reversible (k-1 = 25 and 30/s). Subsequent codon recognition (k2 = 100 and 80/s) stabilizes the complex in an Mg2+-dependent manner (k-2 = 0.2 and 2/s). It induces the GTPase conformation of EF-Tu (k3 = 500 and 55/s), instantaneously followed by GTP hydrolysis. Subsequent steps are independent of Mg2+. The EF-Tu conformation switches from the GTP- to the GDP-bound form (k4 = 60/s), and Phe-tRNAPhe is released from EF-Tu.GDP. The accommodation of Phe-tRNAPhe in the A site (k5 = 8/s) takes place independently of EF-Tu and is followed instantaneously by peptide bond formation. The slowest step is dissociation of EF-Tu.GDP from the ribosome (k6 = 4/s). A characteristic feature of the mechanism is the existence of two conformational rearrangements which limit the rates of the subsequent chemical steps of A-site binding.
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Institute of Molecular Biology, University of Witten/Herdecke, 58448 Witten, Germany.
Wintermeyer
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W
Rodnina
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MV
D003062:Codon; D003198:Computer Simulation; D004926:Escherichia coli; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D010441:Peptide Chain Elongation, Translational; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D011072:Poly U / Q000378:metabolism; D011487:Protein Conformation; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D003062:Codon; D012346:RNA, Transfer, Amino Acyl; D011072:Poly U; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu