title: Initial velocity kinetic analysis of 30 S initiation complex formation in an in vitro translation system derived from Escherichia coli.
abstract: 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.
mesh_terms: D002213:Capsid / Q000096:biosynthesis; D036022:Capsid Proteins; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D007700:Kinetics; D008433:Mathematics; D008715:Methionine / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012358:RNA, Transfer, Met; D016601:RNA-Binding Proteins; D012270:Ribosomes / Q000378:metabolism
chemical_list: D036022:Capsid Proteins; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; D016601:RNA-Binding Proteins; C024988:tRNA, formylmethionine-; D008715:Methionine