Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Computational and Molecular Biophysics

Language

English (en)

Date of Award

January 2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Kathleen Hall

Abstract

Polypyrimidine tract binding protein: PTB) is a highly conserved RNA binding protein comprised of four RRMs: RNA recognition motifs). RRMs are extremely prevalent in all kingdoms of life, and have been very well characterized in terms of structure and RNA binding properties. However, all four RRMs of PTB exhibit unique features that can be exploited to learn more about the RNA selection and binding strategy of PTB, as well more general features involving structure/function relationships and protein unfolding mechanisms. PTB participates in a variety of functions in eukaryotic cells, including alternative splicing, mRNA stabilization, and internal ribosomal entry site: IRES) mediated translation initiation. Its mechanism of RNA recognition is determined in part by the novel geometry of its two C-terminal RNA Recognition Motifs: RRM3 and RRM4), which interact with each other to form a stable complex: PTB1:34). This complex itself is unusual among RRMs, suggesting that it performs a specific function for the protein. In order to understand the advantage it provides to PTB, the fundamental properties of PTB1:34 are examined here as a comparative study of the complex and its two constituent RRMs. Both RRM3 and RRM4 adopt folded, and reasonably stable structures, yet the RNA binding properties of the domains differ dramatically. RRM4 does not bind to RNA, and although RRM3 binds to polypyrimidine tracts, its affinity is significantly weaker than that of PTB1:34. 15N-NMR relaxation experiments show that the interaction between RRM3 and RRM4 induces microsecond motions throughout PTB1:34 and forms a unique RNA binding platform. The motions could be important for RNA selection based on secondary structure, part of an RNA binding mechanism, entropic compensation for formation of PTB1:34, or a mechanism of allosteric communication between binding sites of the RRMs. A mutant protein was designed to address the contribution of the motions to protein function. PTB RRM2 and RRM3 are structurally unique in that they both have a C-terminal extension that adds a fifth -strand to the canonical four stranded -sheet, connected to -strand four by a flexible linker. This extension both extends and occludes the putative RNA binding surface. Other RRM extensions have been reported, and appear to influence protein function through a variety of mechanisms including direct interactions with RNA, participation in protein-protein interactions, or stabilization of the RRM core domain. Studies using a truncated form of RRM3 that lacks the extension show that, in this case, the contribution to protein function is likely due to direct RNA contacts. Finally, PTB RRM4 has a unique chemical melting profile that may be useful for investigating protein unfolding transitions. A tryptophan mutant was engineered to facilitate fluorescence studies, and the protein was found to be a natural 'missing link' between two-state and downhill folders.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7P55KGC

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