Date of Award

Spring 1-1-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Robert T. Batey

Second Advisor

Marcelo C. Sousa

Third Advisor

James A. Goodrich

Abstract

Organisms must be able to respond to small molecule signals to efficiently manage cellular metabolism. The 5' leader sequences of bacterial messenger RNAs contain non coding RNA sequences that are able to bind small molecules and affect genetic regulation. These sequences, termed riboswitches, form the basis of an RNA directed, yet protein independent mechanism through which bacteria can regulate the translation or transcription of genes in response to small molecule metabolites. Since their discovery, a number of RNA riboswitches in complex with their native ligands have been solved by x-ray crystallography. The structures have provided atomic level detail of the intimate interactions each ligand has for the RNA and how ligand binding results in the regulation of gene expression. My thesis extends this work by discussing a family of riboswitches responsible for recognizing one of the largest cofactors found in nature, the cobalamins. Specifically, I characterized a new class of riboswitches responsible for the specific recognition of cobalamin. The structure solution via crystallography shows how this riboswitch recognizes cobalamins in a sequence independent fashion and how the riboswitch utilizes RNA tertiary architecture, rather than changes in secondary structure, to sequester a sequence necessary for the initiation of gene translation in bacteria. Lastly, this thesis describes the methods used for the difficult x-ray structure solution of the adenosylcobalamin riboswitch. With the assistance of multiple heavy atom derivatives, the initial electron density maps were interpreted at 5 Å and eventually extended to the maximum resolution of 3.9 Å . The structure of the 220 nucleotide riboswitch shows how 5'-deoxyadenosylcobalamin is specifically recognized via a non-canonical base pair with one of the most conserved sequence elements within the riboswitch, supported by a highly organized RNA architecture and long-range tertiary interactions. Both structures show how two distinct classes of cobalamin riboswitches can recognize their ligands. The results of this thesis benefit a burgeoning community focused on developing therapeutic agents targeting these RNA elements and have broad implications for RNA mediated gene regulation by small molecules.

Included in

Biochemistry Commons

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