Date of Award

Spring 1-1-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Robert T. Batey

Second Advisor

Marcelo C. Sousa

Third Advisor

Roy Parker

Fourth Advisor

James A. Goodrich

Fifth Advisor

Norman R. Pace

Abstract

The scientific discipline known as synthetic biology aims to develop a set of tools and engineering principles to design artificial biological systems for the production of commodity chemicals like biofuels and therapeutics. A variety of bacterial non-coding RNAs named riboswitches, are particularly attractive for the field of synthetic biology for their ability to create intricate tertiary structures capable of binding a small molecule metabolite and translating this event into the regulation of gene expression. Riboswitches have been studied with a variety of biophysical and biochemical techniques that have provided a wealth of information useful in the rational design of synthetic RNA systems. However, although the field has been successful in creating artificial riboswitches that work robustly in vitro, when these RNAs are expressed in the cellular environment they fail to achieve their purpose. I believe that the reason for these failures originates from the lack of research efforts that aimed to understand riboswitches in the intracellular environment.

To better understand the in vivo regulatory activity of riboswitches, I designed an intracellular reporter assay that couples the riboswitch's activity to a quantifiable fluorescence output. With this fluorescence reporter assay, I performed a comprehensive mutagenesis analysis of the Bacillus subtilis adenine-binding pbuE riboswitch, probing the structural features identified as being responsible for ligand binding, global structure acquisition, and regulation of transcription. The results of this mutational analysis revealed that some of the structural motifs, for example a conserved loop-loop interaction that is important for ligand binding in vitro, display differential effects in their contribution to the in vivo regulatory activity. Moreover, the intracellular assay allowed me to evaluate structural elements of the expression platform that direct the regulatory activity, an aspect of riboswitches that has been understudied. My data revealed that the stability of a nucleator stem-loop element at the distal tip of the transcriptional terminator stem has a significant impact on the regulatory activity, most likely affecting the rate of the terminator formation. With this new data, I proposed a mechanism of regulation that suggests that the ligand interaction with the aptamer acts as a kinetic barrier between the interchange of the alternative secondary structures that mediate the riboswitch's regulatory response.

With the new insights of the in vivo activity of the purine riboswitch, I describe the creation of innovative techniques towards the development of novel synthetic riboswitches. This new approach attempts to create an artificial evolution system, in which using the tetracycline antibiotic resistance marker, I can select for riboswitches that bind novel ligands and capable of producing a regulatory response. The preliminary results of these experiments indicate that riboswitches are a robust platform for the in vivo directed evolution of RNAs, and that with the results generated in the purine riboswitch in vivo mutational analysis, I can provide ideas to improve the selection platform. With the development of this technology, I attempt to contribute to the discipline of synthetic biology by providing novel RNA tools that will enable the future of biotechnology.

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