Mutagenesis of a stacking contact in the MS2 coat protein-RNA complex

EMBO J. 1996 Dec 16;15(24):6847-53.

Abstract

The thermodynamic contribution of a stacking interaction between Tyr85 in MS2 coat protein and a single-stranded pyrimidine in its RNA binding 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 photocrosslinks with iodouracil-containing RNA at the same rate as the wild-type protein, the mutant proteins interact with RNA in a similar manner. The pH dependence of KD for the Phe and His proteins differs substantially from the wild-type protein, suggesting that the titration of position 85 contributes substantially to the binding properties. Experiments with specifically substituted phosphorothioate RNAs confirm a hydrogen bond between the hydroxyl group of tyrosine and a phosphate predicted by the crystal structure.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Capsid / chemistry*
  • Capsid Proteins*
  • Cloning, Molecular
  • Hydrogen-Ion Concentration
  • Molecular Structure
  • Mutagenesis
  • Photochemistry
  • RNA / chemistry*
  • RNA Probes
  • RNA-Binding Proteins / chemistry*
  • Thermodynamics

Substances

  • Capsid Proteins
  • RNA Probes
  • RNA-Binding Proteins
  • RNA