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J Biol Chem. 1984 Jun 25;259(12):7607-14.

Initial velocity kinetic analysis of 30 S initiation complex formation in an in vitro translation system derived from Escherichia coli.


The analysis of initial velocity kinetic data was used to examine the order in which fMet-tRNA and the coat cistron of genomic bacteriophage R17 or Q beta RNA bind to the 30 S ribosome subunit. These data were obtained using a quantitative assay for protein synthesis in Escherichia coli extracts where the rate of accumulation of protein product is dependent on the concentration of mRNA and is partially dependent on fMet-tRNA. Under the conditions of this assay, the amount of protein synthesized was proportional to the formation of ternary complexes between the mRNA, fMet-tRNA, and the 30 S ribosomal subunit. The results from the initial velocity and alternative substrate experiments are consistent with a rapid equilibrium ordered mechanism as opposed to a rapid equilibrium random mechanism. Analysis of the rate of coat protein synthesis at varied concentrations of mRNA and fixed concentrations of fMet-tRNA indicated that fMet-tRNA was the first substrate to bind to the 30 S subunit when either coat cistron was used as the mRNA. This scheme assumes the existence of a relatively slow step in protein synthesis that occurs after both the initiating tRNA and mRNA are bound to the ribosome and which allows substrate addition to reach thermodynamic equilibrium.

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