Understanding Synthetic Riboswitch Mechanism and Creating Predictive Biophysical Model for Designing Novel Synthetic Riboswitches Open Access

Wang, Jingzhi (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/gh93gz99q?locale=en


As an emerging class of genetic regulatory elements, synthetic riboswitch has become a handy tool that allows conditional regulation of gene expression and facilitates the downstream application with great versatility. Previous efforts have successfully expanded the repertoire of synthetic riboswitches; however, the knowledge of synthetic riboswitch mechanism remains unclear. In this thesis, we present a detailed molecular study to reveal mechanistic insights of our model theophylline riboswitch. This study helps to construct the first biophysical model of synthetic riboswitch that has predictive power. Meanwhile, the kinetic aspect of riboswitch mechanism is analyzed to support the hypothesis that riboswitch function contains both thermodynamic and kinetic components. Furthermore, we implement our biophysical model to design a novel class of synthetic tetramethylrosamine riboswitches. Detailed characterization of these riboswitches is provided to illustrate their mechanism and guide further applications. Each chapter reveals one aspect of riboswitch function and advances our ability to create more efficient synthetic riboswitches.

Table of Contents

Chapter 1 - Introduction. 1

1.1 Genetic Regulators and Riboswitches. 1

1.2 The Discovery of Naturally Occurring Riboswitches and Their General Characteristics. 3

1.3 Prevalence of Riboswitches Supports the RNA World Hypothesis. 4

1.4 Synthetic Riboswitches in Gene Regulation. 9

1.4.1 The Rationale behind Engineering Riboswitches. 9

1.4.2 Methods for Developing Synthetic Riboswitches. 11

1.4.3 Different Approaches to Design Synthetic Riboswitches. 14

1.4.4 The Performance of Riboswitch in Model Species. 19

1.5 Different Types of Riboswitch Mechanisms. 21

1.6 Conclusion. 25

1.7 Reference. 26

Chapter 2 Investigation into the Mechanism of Theophylline Riboswitches. 33

2.1 Introduction. 33

2.2 Results. 38

2.2.1 Mutation Studies Reveal Riboswitch Mechanistic Insight. 38

2.2.2 Characterization of Direct Interaction between RNA Molecule and Theophylline Using In-line Probing Assay. 41

2.2.3 Construction of Detailed Biophysical Model for Theophylline Riboswitches. 45

2.3 Discussion. 47

2.4 Experimental. 54

2.5 References. 59

Chapter 3 Studying Kinetic Aspects of the mechanism of a Theophylline Riboswitch. 61

3.1 Introduction. 61

3.2 Results. 66

3.2.1 Time Dependence of Theophylline Riboswitch Function. 66

3.2.2 Investigation to the influence of ligand binding in RNA stability. 69

3.2.3 In vivo Transcription Translation Decoupling Experiment. 71

3.3 Discussion. 74

3.4 Experimental. 79

3.5 References. 83

Chapter 4 Computer-assisted Screening for Tetramethylrosamine Riboswitches. 85

4.1 Introduction. 85

4.2 Results.91

4.2.1 In vitro Characterization of Novel Tetramethylrosamine Riboswitches Predicted by Computational Methods. 91

4.2.2 In vivo Characterization of Novel Tetramethylrosamine Riboswitches Predicted by Computational Methods. 92

4.2.3 In vitro Transcription-Translation Decoupling Experiment of Novel Tetramethylrosamine Riboswitches. 97

4.2.4 In vitro and In vivo Characterization of Novel Tetramethylrosamine Riboswitches with Different DNA Template Concentration. 99

4.3 Discussion. 101

4.4 Experimental. 107

4.5 References. 113

Chapter 5 - Conclusions. 116

5.1 Summaries and Conclusions. 116

5.2 References. 119

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