Genetic Analysis of a Translational Repressor

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DESCRIPTION (provided by applicant): Specific recognition of RNAs by proteins is fundamental to a wide variety of biological processes. For example, a number of steps in gene expression, including RNA-splicing and protein synthesis depend on extensive interactions of RNAs and proteins. Moreover, RNA-protein recognition plays an essential role in the life cycles of important viral pathogens, including the Human Immunodeficiency and Hepatitis C Viruses, to name only two. Many of the proteins that bind RNA do so using a beta-sheet. In fact, we now recognize the existence of whole classes of related proteins that have been adapted by mutation to the binding of an amazing diversity of RNAs on structurally homologous beta-sheets. The coat proteins of the RNA bacteriophages offer an unusually accessible experimental model for studying this form of RNA recognition. Since related viruses use coat proteins with the same tertiary fold to bind different RNAs, they provide the opportunity to explore how variation in amino acid sequence alters the specificity of the RNA-binding site. The best understood example is the MS2 coat protein-RNA complex, which has been intensively studied using both functional and structural approaches. This application seeks to build on that knowledge by working toward a similarly detailed understanding of several evolutionarily variant coat proteins with divergent RNA-binding specificities. Also proposed are experiments designed to probe the limits of adaptability of the coat protein beta-sheet through the deliberate evolution of new RNA-binding activities on the MS2 structural framework. Understanding how coat protein can be adapted to the binding of diverse RNAs should illuminate principles that govern the recognition of RNA by beta-sheets generally.