Date of Award

Spring 1-1-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Robert T. Batey

Second Advisor

Dylan J. Taatjes

Third Advisor

Thomas Blumenthal

Fourth Advisor

Shelley D. Copley

Fifth Advisor

Marcelo C. Sousa

Abstract

Riboswitches are structural elements of gene control located in the 5’- untranslated region of some mRNAs capable of regulating the expression of important genes. The mode of regulation occurs by binding a cellular metabolite if its concentration excess a threshold and controlling genes involved in the biogenesis or transport of this ligand. A common riboswitch is composed of two functionally distinct units: i) the aptamer domain, which recognizes and binds the cellular metabolite and ii) an expression platform responsible for interaction with the cellular machinery and control of gene production. The aptamer domain of riboswitches confers high degree of affinity and specificity towards the correct ligand. This binding event rearranges the secondary structure of the expression platform, which in turn allows regulation of gene expression. Riboswitch genetic control is observed in the three domains of life. However, it is most important in bacteria.

Previously, the characterization of riboswitches has focused on the biophysical study of different aptamer domains and the changes that occur when the ligand binds the RNA. These studies have revealed the intricate recognition of essentially every functional group of a diverse set of cellular metabolites by RNAs. However, the signal transduction from the aptamer domain to the expression platform remains uncharacterized. Most importantly, the secondary structural switch that an expression platform undergoes has been reduced to a simple variation of secondary interactions. In this dissertation, I have functionally characterized, both in vitro and in vivo, two different riboswitches in their entirety, demonstrating novel findings about the regulatory response of these riboregulators. The results show that small secondary structures formed within the expression platform are important for gene control and that the ability of the switching mechanism is innate within this domain (independent of ligand presence).

The application of these learned principles of riboswitch regulation for the development of novel RNA-based biosensors has also been accomplished. To establish engineering strategies capable of developing general, reliable, and modular RNA devices, I mixed and matched natural and SELEX-derived (systematic evolution of ligands by exponential enrichment) aptamers with a variation of natural expression platforms. These engineered riboswitches are functional in vitro and in E. coli, and present a new avenue to control cellular regulation for different applications including synthetic biology.

Share

COinS