Date of Award

Spring 1-1-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Robert T. Batey

Second Advisor

Marcelo C. Sousa

Third Advisor

Dylan J. Taatjes

Fourth Advisor

Amy E. Palmer

Fifth Advisor

Thomas T. Perkins

Abstract

RNA-based genetic regulatory mechanisms in bacteria are critical for maintaining normal cellular homeostasis and response to a broad spectrum of environmental and intracellular signals. Riboswitches are structured elements typically found in the 5' leader regions of mRNAs that often control the expression of genes involved in the transport or biosynthesis of small-molecule metabolites. These regulatory elements are comprised of two domains, a highly conserved receptor (aptamer domain) that directly binds a small-molecule and a regulatory domain (expression platform) that controls the gene expression machinery.

Atomic level structures of riboswitch receptor domains bound to their effector have revealed how mRNAs recognize small-molecules, but mechanistic details linking the structures to gene regulation remain elusive. To address this, I helped solve crystal structures of two different classes (class-I and class-II) of cobalamin riboswitches that include the regulatory element of the downstream expression platform. These structures reveal a composite cobalamin-RNA scaffold stabilizes an unusual long-range intramolecular kissing-loop interaction that controls gene expression via occlusion of the ribosome binding site. A combination of cell-based, biochemical, and biophysical techniques further defines the specific structural features that dictate the stability and kinetics of cobalamin-dependent kissing-loop assembly. These data demonstrate the rate of kissing-loop formation directs a regulatory response that couples translation initiation with general mechanisms that control mRNA abundance.

The crystal structures also reveal the two classes share a common cobalamin-binding core, but use distinct peripheral extensions to recognize different B12 derivatives. In each case, recognition is accomplished through shape complementarity between the RNA and cobalamin, with relatively few hydrogen bonding interactions that typically govern RNA-small molecule binding. Unexpectedly, structurally similar members of class-II riboswitches display a broad range of binding affinities for different cobalamin variants. Systematic mutagenesis coupled with calorimetric binding data show that selectivity for cobalamins is dictated by interactions between a peripheral element of the RNA with a region of the binding core that makes direct contacts with the ligand. These data are consistent with a binding mechanism that uses flexibility within single stranded regions of the RNA to either exclude or accommodate cobalamin derivatives based on size and steric constraints.

Included in

Biochemistry Commons

Share

COinS