Prokaryotic initiation factor 2 acts at the level of the 30 S ribosomal subunit A fluorescence stopped-flow study

AbstractThe mechanism of action of initiation factor 2 (IF2) from Escherichia coli during initiation of translation was investigated by kinetic analysis of the binding of N-AcPhe-tRNAphe or Phe-tRNAphe to poly(U)-programmed 30 S ribosomal subunits. The reaction was studied by using the stopped-flow technique, monitoring the fluorescence signal of a proflavine inserted next to the anticodon (position 37) of yeast tRNAphe. Both the rate and extent of N-AcPhe-tRNAphe binding to 30 S subunits are strongly enhanced by IF2. The effect of IF2 was studied at different stoichiometric ratios between factor, ribosomes, and N-AcPhe-tRNAphe, in both the presence and absence of the other two factors. In all cases, the IF2 effect titrates with the 30 S ribosomes. This is also the case in the presence of an equimolar amount of 50 S ribosomal subunits. Furthermore, IF2 was found to stimulate strongly the binding of Phe-tRNAphe, in spite of the inability of the latter to form a detectable binary complex with IF2. The results are interpreted to mean that IF2 acts in a stoichiometric rather than a catalytic fashion, at the level of the 30 S ribosomal subunit. They do not support a model in which IF2 acts as a carrier for the initiator tRNA