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 bacteriophag e MS2 coat protein subunits and synthetic RNA fragments encompassing the na tural translational operator site, or the consensus sequences of three dist inct RNA aptamer families, which are known to bind to the same site on the protein. Reactions were assayed using stopped-flow fluorescence spectroscop y and either the intrinsic tryptophan fluorescence of the protein or the si gnals from RNA fragments site-specifically substituted with the fluorescent adenosine analogue 2'-deoxy, 2-aminopurine. The kinetics observed were ind ependent of the fluorophore being monitored or its position within the comp lex, 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 i s at least biphasic. The faster phase, constituting 80-85 % of the amplitud e, is a largely diffusion driven RNA-protein interaction (k(1) approximate to 2 x 10(9) M-1 s(-1)). The salt dependence of the forward reaction and th e similarities of the on-rates of lower-affinity RNA fragments are consiste nt with a diffusion-controlled step dominated by electrostatic steering. Th e slower phase is independent of reactant concentration, and appears to cor respond to isomerisation of the coat protein subunit(s) prior to RNA bindin g (k(iso) approximate to 0.23 s(-1)). Measurements with a coat protein muta nt (Pro78Asn) show that this phase is not due to cis-tuans isomerisation at this residue. The conformational changes in the protein ligand during form ation of an RNA-protein complex might play a role in the triggering of caps id self-assembly and a model for this is discussed. (C) 2001 Academic Press .