Modifying the specificity of an RNA backbone contact.
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.
Dertinger
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Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA.
Dale
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Uhlenbeck
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D001665:Binding Sites; D002213:Capsid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D003855:Deoxyribose / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D017909:Levivirus / Q000737:chemistry* / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D015202:Protein Engineering; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012266:Ribose / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D036022:Capsid Proteins; D016601:RNA-Binding Proteins; D018698:Glutamic Acid; D003855:Deoxyribose; D012313:RNA; D012266:Ribose