Date of Award

Spring 1-1-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Deborah S. Wuttke

Second Advisor

Robert T. Batey

Third Advisor

Roy Parker

Fourth Advisor

Robert D. Kuchta

Fifth Advisor

Loren E. Hough

Abstract

Protein-ligand specificity forms the fundamental basis for many biological mechanisms with properly tuned binding being required for most biological processes. Aberrant interactions can result in consequences ranging from wasted cellular resources to disease pathologies and death. As such, characterizing interaction specificities is a critical step in understanding biological systems. In this thesis I have characterized the RNA-binding properties of the telomere protection protein Pot1, developed an optimized SELEX protocol to characterize RNA-binding by newly identified RNA-binding proteins, and expanded on the RNA-binding specificity of one of those proteins, the epigenetic regulator CypE.

High fidelity binding to ssDNA, but not ssRNA, is integral to the function of the essential telomere end protection protein Pot1, In S. pombe, this presents a unique challenge as the C-terminal domain of the DNA-binding domain, Pot1pC, exhibits non-specific ssDNA recognition, achieved through thermodynamically equivalent alternative binding conformations. Given this malleability, how simultaneous specificity for ssDNA over RNA is achieved was unclear. Examination of the ribose-position specificity of Pot1pC shows that ssDNA specificity is additive but not uniformly distributed across the ligand. High-resolution structures of Pot1pC in complex with RNA-DNA chimeric ligands reveal Pot1pC discriminates against RNA by utilizing conserved non-compensatory binding modes that feature significant rearrangement of the binding interface. These alternative conformations, accessed through both ligand and protein flexibility, recover much, but not all, of the binding energy, leading to the observed reduction in affinities suggesting that intermolecular interfaces are remarkably sophisticated in their tuning of specificity towards flexible ligands.

Recent discovery of widespread RNA-binding by unexpected RNA binders highlights the need for functional characterization of these non-canonical RNA-binding domains. SELEX, combined with new sequencing technologies, represents an ideal technique to do this. Using CypE, an RNA-binding cyclophilin involved in splicing and chromatin remodeling, I have optimized a selection protocol for other cyclophilins. Selection against CypE, while not identifying an RNA that binds the cyclophilin, reveals an aptamer with 20-fold tighter binding than previously reported with an extended binding interface on the RRM, suggesting RNA as a competitive ligand for CypE and provoking implications for the role of RNA in CypE gene repression.

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