Modifying the specificity of an RNA backbone contact

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.
• 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.