Date of Award

Spring 1-1-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Robert T. Batey

Second Advisor

James A. Goodrich

Third Advisor

Marcelo C. Sousa

Fourth Advisor

Roy Parker

Fifth Advisor

Norm R. Pace

Abstract

RNA is a macromolecule capable of a diverse set of roles within a cell. In addition to acting as a messenger to span the informational gap between DNA and protein, RNA has been shown to regulate gene expression in both bacteria and eukaryotes. In this thesis I explore two classes of RNA regulatory elements found in bacteria called riboswitches. These sequences are highly structured regions within the 5' leader sequence of bacterial mRNAs that control gene expression through direct interaction between a small molecule metabolite and the RNA.

The tetrahydrofolate (THF) riboswitch is a conserved RNA element proposed to regulate gene expression based on the presence of THF, while discriminating against closely related analogs. To understand how THF binding directs gene expression, I solved the crystal structure of the THF riboswitch receptor domain in complex with THF. The structure revealed two THF molecules bound to a single structured RNA that display cooperative binding under physiological conditions. This is the first time a single RNA receptor domain (natural or in vitro selected) has been observed to bind two ligands. Screening of closely related analogs revealed that the para-aminobenzoic acid (pABA) moiety of THF is important for regulation, despite playing a minor role in binding affinity. I also found an alternative binding mode of adenine and related purine compounds that may have important implications in small molecule targeting of RNA.

The second RNA studied was the SAM-I/IV riboswitch. This RNA shares a common ligand binding core with other S-adenosylmethionine (SAM) sensing riboswitches (SAM-I, SAM-IV), but has a radically divergent peripheral architecture. The crystal structure revealed this alternative peripheral architecture is critical for both high affinity SAM recognition and regulation of mRNA translation. Further, I demonstrate the stability of a pseudoknot found in the SAM-I/IV and SAM-IV riboswitches is strongly linked to the efficiency of gene repression in vivo. Together, these data provide a strong mechanistic basis for the linkage between SAM binding and regulation of gene expression.

The work presented here represents a significant contribution to our understanding of riboswitch structure and function, and more generally, our knowledge of RNA-based regulation.

Included in

Biochemistry Commons

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