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Complete kinetic mechanism of elongation factor Tu‐dependent binding of aminoacyl‐tRNA to the A site of the E.coli ribosome
Author(s) -
Pape Tillmann,
Wintermeyer Wolfgang,
Rodnina Marina V.
Publication year - 1998
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/17.24.7490
Subject(s) - biology , ef tu , ribosome , elongation factor , aminoacyl trna , mechanism (biology) , transfer rna , escherichia coli , translation (biology) , protein biosynthesis , a site , binding site , elongation , biochemistry , microbiology and biotechnology , rna , messenger rna , gene , physics , materials science , ultimate tensile strength , metallurgy , quantum mechanics
The kinetic mechanism of elongation factor Tu (EF‐Tu)‐dependent binding of Phe‐tRNA Phe to the A site of poly(U)‐programed 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‐tRNA Phe is rapid ( k 1 = 110 and 60/μM/s at 10 and 5 mM Mg 2+ , 20°C) and readily reversible ( k −1 = 25 and 30/s). Subsequent codon recognition ( k 2 = 100 and 80/s) stabilizes the complex in an Mg 2+ ‐dependent manner ( k −2 = 0.2 and 2/s). It induces the GTPase conformation of EF‐Tu ( k 3 = 500 and 55/s), instantaneously followed by GTP hydrolysis. Subsequent steps are independent of Mg 2+ . The EF‐Tu conformation switches from the GTP‐ to the GDP‐bound form ( k 4 = 60/s), and Phe‐tRNA Phe is released from EF‐Tu·GDP. The accommodation of Phe‐tRNA Phe in the A site ( k 5 = 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 ( k 6 = 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.