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

Karen A. LeCuyer, Linda S. Behlen, Olke C. Uhlenbeck*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

49 Scopus citations

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 K(D) 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.

Original languageEnglish (US)
Pages (from-to)6847-6853
Number of pages7
JournalEMBO Journal
Volume15
Issue number24
DOIs
StatePublished - 1996

Keywords

  • Phosphorothioates
  • Photocrosslinking
  • RNA-protein interactions

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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