Modifying the specificity of an RNA backbone contact

Dagmar Dertinger, Taraka Dale, Olke C. Uhlenbeck

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

The interaction between the MS2 bacteriophage coat protein homodimer and its cognate RNA hairpin is facilitated by 21 different RNA-protein contacts. In one of these contacts, the 2′-hydroxyl group at ribose -5 of the RNA acts as a hydrogen bond donor to Glu63 in one subunit of the protein. Previous experiments showed that substitution of ribose -5 with deoxyribose resulted in a 24-fold decrease in binding affinity between RNA and protein. Using a protein where the two MS2 monomers were fused to increase stability, the contribution of this contact to the overall binding affinity was investigated by site-directed mutagenesis. When Glu63 was substituted with glutamine, aspartate, or alanine, the binding affinity of the hairpin for the protein was weakened by 12 to 100-fold, similar to that observed with deoxyribose at position -5. However, the specificity of the three mutant proteins for RNAs with various modifications at the 2′-position of ribose -5 differed dramatically. While the Glu63Asp protein resembled the wild-type protein in preferring the 2′-hydroxyl group over a proton or a bulky 2′-substituent, both the Glu63Ala and Glu63Gln proteins preferred bulky 2′-substituents over the 2′-hydroxyl group by more than 100-fold. These experiments emphasize the ease with which the specificity of a protein-nucleic acid interaction can be changed at thermodynamically important sites.

Original languageEnglish (US)
Pages (from-to)649-654
Number of pages6
JournalJournal of Molecular Biology
Volume314
Issue number4
DOIs
StatePublished - Dec 11 2001

Keywords

  • 2′-hydroxyl substitutions
  • MS2 coat protein
  • Phosphorothioate linkage
  • RNA-binding protein

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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