MUTANTS OF THE BACTERIOPHAGE-MS2 COAT PROTEIN THAT ALTER ITS COOPERATIVE BINDING TO RNA

An RNA binding assay measuring cooperative protein binding has been us ed to evaluate the effects of mutations in the MS2 phage coat protein expected to disrupt capsid assembly. By using the crystal structure of the virus as a guide, six different mutations in the FG loop structur e were selected in which hydrophobic residues were replaced with charg ed residues. Most of these proteins form capsids in Escherichia coli, but not in an in vitro assembly assay, suggesting that interdimer inte ractions are weaker than wild type. These mutant proteins reduce the f ree energy of cooperative protein binding to a double-hairpin RNA from its wild-type value of -1.9 kcal/mol. Several of the variants that ha ve large effects on cooperativity have no effect on RNA affinity, sugg esting that protein-RNA interactions can be affected independently of dimer-dimer interactions. The V75E;A81G protein, which shows no measur able cooperativity, binds operator RNA equally well as the wild-type p rotein under a variety of buffer conditions. Because this protein also exhibits similar specificity for variant RNA sequences, it will be us eful for studying RNA binding properties independent of capsid assembl y.