Effect of Escherichia coli initiation factors on the kinetics of N-AcPhe-tRNAPhe binding to 30S ribosomal subunits. A fluorescence stopped-flow study

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Abstract
The mechanism of binding of N-AcPhe-tRNAPhe (yeast) to poly(U)-programmed E. coli 30S ribosomal subunits and the effect of individual initiation factors (IF-1, IF-2 and IF-3) and GTP on this process was studied by fluorescence stopped-flow kinetic measurements. The formation of the ternary complex was followed by an increase of intensity and polarization of the fluorescence of a proflavin label located in the anticodon loop of the tRNA. The effect of the initiation factors and GTP is to increase the velocity of ternary complex formation (about 400-fold at 7 mM Mg2+). In the presence of the 3 initiation factors and GTP the formation of the ternary complex could be resolved into 2 partial reactions: a fast apparently second-order step, followed by a slow rearrangement step. The data suggest a mechanism in which the ternary complex is formed by at least 2 rearrangements of an initially formed preternary complex. The accelerating effects of IF-2 and IF-3 can be understood by assuming a synergistic allosteric action of the factors on the 30S ribosomal subunit, whereas IF-1 appears to act indirectly by influencing the other 2 factors.