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Related Experiment Videos

Modifying the specificity of an RNA backbone contact.

D Dertinger1, T Dale, O C Uhlenbeck

  • 1Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA.

Journal of Molecular Biology
|December 6, 2001
PubMed
Summary
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Investigating the MS2 bacteriophage coat protein and RNA hairpin interaction, researchers found that altering a key residue (Glu63) significantly impacts binding affinity and specificity. This highlights the flexibility of protein-nucleic acid interactions.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Structural Biology

Background:

  • The MS2 bacteriophage coat protein (MCP) interacts with its cognate RNA hairpin through multiple RNA-protein contacts.
  • A specific contact involves the RNA's ribose 2'-hydroxyl group and a glutamate residue (Glu63) in the protein, crucial for binding affinity.

Purpose of the Study:

  • To investigate the contribution of the RNA 2'-hydroxyl and protein Glu63 contact to the binding affinity and specificity of the MCP-RNA interaction.
  • To understand how mutations at Glu63 affect the protein's preference for RNA modifications.

Main Methods:

  • Site-directed mutagenesis was used to substitute Glu63 with glutamine, aspartate, or alanine in a stabilized MCP homodimer.
  • Binding affinities of mutant proteins for the RNA hairpin were measured.

Related Experiment Videos

  • Specificity of mutant proteins for RNAs with various 2'-position modifications was assessed.

    • Main Results:

    • Mutations at Glu63 (to Gln, Asp, Ala) reduced binding affinity by 12-100 fold, similar to deoxyribose substitution.
    • Glu63Asp mutant retained wild-type specificity for the 2'-hydroxyl group.
    • Glu63Ala and Glu63Gln mutants showed a >100-fold preference for bulky 2'-substituents over the 2'-hydroxyl group.

    Conclusions:

    • The Glu63-RNA 2'-hydroxyl contact is thermodynamically important for MCP-RNA binding affinity.
    • Specific mutations at Glu63 can dramatically alter the protein's specificity for RNA modifications at the 2'-position.
    • Protein-nucleic acid interaction specificity can be readily modulated at key contact sites.