Kinetics of ribosome dissociation and subunit association. The role of initiation factor IF3 as an effector.

The association-dissociation kinetics of Escherichia coli MRE 600 "tight" (Type A) ribosomes (30 S + 50 S e 70 S ) for a variety of conditions have been studied in a light-scattering stopped-flow apparatus.The rate constants for the M&'-induced subunit association and 70 S dissociation reactions were 1.7 X lo' M" s-' (18 n m M&') and 0.015 s-l (1 m M@'), respectively.The addition of ribosomal protein SI had no effect on either reaction, whereas incubation of ribosomal subunits with initiation factor 3 reduced the M&'-induced association rate by a factor of five.Two further types of experiments were carried out with IF3.In the first experiment, ribosomal subunits were flowed against both IF3 and M@', resulting in a rate identical with that obtained in the absence of IF3.The second experiment was a time-lapsed stopped-flow experiment in which ribosomal subunits were incubated for various times with IF3 and then flowed against Mg'.The results of these experiments were incompatible with, the predictions of the anti-association model which assumes that the 30 S-1F3 complex is incapable of forming 70 S ribosomes.In terms of a simple model, we obtain a rate constant of 6.2 X lo3 M" s-' for IF3 combination with 30 S subunits at 1 m M Mg2+.The spermidine-induced association reaction w a s monophasic at 10 m spermidine with a rate constant identical with that found for M&'-induced association.At lower spermidine concentrations (5 and 2.5 m), the data could best be described as biphasic.An allosteric model is proposed to account for the kinetic results for spermidine and M&'-induced association, subunit dissociation, and the effects of 1F3.This model proposes two conformations for both the subunits and the 70 S ribosome.