Kinetic studies on the interaction of chain initiation factor 3 with 70 S Escherichia coli ribosomes and subunits.

The magnesium ion dependencies of several of the reaction rates for assembly of the Escherichia coli protein synthesis initiation complex have been determined as well as the effects of IF-3 (initiation factor 3) on the overall reaction of 30 S and 50 S ribosomal subunits. The reaction kinetics were studied by light-scattering changes as well as by changes in the fluorescence anisotropy of dansylated-IF-3. The full model for treating these processes consists of four reactions, three of which are thermodynamically independent. Conditions were chosen and techniques were employed that allowed three of the reactions to be studied individually. This allowed us to fit, with a single adjustable rate constant, all of the light-scattering changes that occurred upon flowing 30 S and 50 S ribosomal subunits against varying concentrations of both Mg2+ and IF-3. Preparations of IF-3 were found to react toward 30 S subunits with either of two markedly different binding rates. We find that the simplest model that explains both the light-scattering and anisotropy data for all IF-3 experiments is one that includes as a necessary step the association of 30 S-IF-3 with 50 S subunits to form a 70 S-IF-3 complex.