MUTAGENESIS OF A STACKING CONTACT IN THE MS2 COAT PROTEIN-RNA COMPLEX

The thermodynamic contribution of a stacking interaction between Tyr85 in MS2 coat protein and a single-stranded pyrimidine in its RNA bindi ng site has been examined, Mutation of Tyr85 to Phe, His, Cys, Ser and Ala decreased the RNA affinity by 1-3 kcal/mol under standard binding conditions, Since the Phe, His and Cys 85 proteins formed UV photocro sslinks with iodouracil-containing RNA at the same rate as the wild-ty pe protein, the mutant proteins interact with RNA in a similar manner. The pH dependence of K-D for the Phe and His proteins differs substan tially from the wild-type protein, suggesting that the titration of po sition 85 contributes substantially to the binding properties, Experim ents with specifically substituted phosphorothioate RNAs confirm a hyd rogen bond between the hydroxyl group of tyrosine and a phosphate pred icted by the crystal structure.