Probing the kinetics of formation of the bacteriophage MS2 translational operator complex: identification of a protein conformer unable to bind RNA.

We have investigated the kinetics of complex formation between bacteriophage MS2 coat protein subunits and synthetic RNA fragments encompassing the natural translational operator site, or the consensus sequences of three distinct RNA aptamer families, which are known to bind to the same site on the protein. Reactions were assayed using stopped-flow fluorescence spectroscopy and either the intrinsic tryptophan fluorescence of the protein or the signals from RNA fragments site-specifically substituted with the fluorescent adenosine analogue 2'-deoxy, 2-aminopurine. The kinetics observed were independent of the fluorophore being monitored or its position within the complex, indicating that the data report global events occurring during complex formation. Competition assays show that the complex being formed consists of a single coat protein dimer and one RNA molecule. The binding reaction is at least biphasic. The faster phase, constituting 80-85 % of the amplitude, is a largely diffusion driven RNA-protein interaction (k1 approximately 2x10(9) M(-1) s(-1)). The salt dependence of the forward reaction and the similarities of the on-rates of lower-affinity RNA fragments are consistent with a diffusion-controlled step dominated by electrostatic steering. The slower phase is independent of reactant concentration, and appears to correspond to isomerisation of the coat protein subunit(s) prior to RNA binding (k(iso) approximately 0.23 s(-1)). Measurements with a coat protein mutant (Pro78Asn) show that this phase is not due to cis-trans isomerisation at this residue. The conformational changes in the protein ligand during formation of an RNA-protein complex might play a role in the triggering of capsid self-assembly and a model for this is discussed.