Molecular characterization of ribosome-independent toxin substrate specificity Open Access

Hoffer, Eric David (2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/8336h240k?locale=en
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Abstract

Bacteria have a remarkable ability to survive rapidly fluctuating environments such as nutrient deprivation and antibiotic treatment. There are several genes and pathways that contribute to bacterial survival during stress and one of the most abundant are the two-component systems known as toxin-antitoxins. Despite the nomenclature, toxin-antitoxins are beneficial to their host bacterium and are associated with the formation of non-growing, highly stress tolerance cells known as persisters. The toxin inhibits cell growth through disrupting essential cellular processes and the antitoxin inhibits the toxin under non-stress conditions. The work presented here describes the structure and substrate specificity of two toxin families. The first is from the VapC toxin family known as VapC-mt4 from Mycobacterium tuberculosis and was found to cleave elongator tRNAs t0 downregulate protein synthesis. To better understand how VapC recognizes tRNAs, I generated a homology model and simulated binding to tRNA and found that VapC may have different modes of binding depending on the length of substrate tRNA variable loop. In this study I also determined the structure and characterized the active site of MazF-mt6, a member of the CcdB/MazF toxin family that has an unusual specificity for ribosomal RNA. Through this research I show that MazF-mt6 has an irregular β1-β2 linker that likely plays a role in allowing structured RNA to enter the active site. Finally, I attempted to solve the crystal structures of S. flexneri VapC and Mtb MazF-mt9 bound to substrate tRNA. Though I was unable to solve the target crystal structure, I was able to optimize the purification of both toxins and tRNA substrates and show that the VapCD7A variant was able to stably bind tRNAfMet. These different research projects have helped to expand our understanding of bacterial toxin structure and substrate specificity.

Table of Contents

Table of Contents

Chapter 1: Introduction .................................................................................................... 1

Challenges of antibiotic tolerance.......................................................................................... 1

Bacterial stringent response................................................................................................. 2

Classification of toxin-antitoxin systems.................................................................................. 3

Type II toxin-antitoxin systems............................................................................................. 3

MazEF toxin-antitoxin system................................................................................................ 5

VapBC toxin-antitoxin system................................................................................................ 7

Questions addressed........................................................................................................... 8

Figures............................................................................................................................ 10

References....................................................................................................................... 14

Chapter 2: Growth-regulating Mycobacterium tuberculosis VapC-mt4 toxin

is an isoacceptor-specific tRNase...................................................................................... 18

Abstract........................................................................................................................... 20

Introduction...................................................................................................................... 21

Results............................................................................................................................. 23

Discussion......................................................................................................................... 30

Methods........................................................................................................................... 36

Figures............................................................................................................................. 44

References........................................................................................................................ 67

Chapter 3: The structure and function of Mycobacterium tuberculosis MazF-mt6

provides insights into conserved features of MazF endonucleases........................................ 71

Abstract............................................................................................................................ 72

Introduction....................................................................................................................... 73

Results.............................................................................................................................. 74

Discussion.......................................................................................................................... 79

Experimental procedures........................................................................................................ 83

Figures and tables................................................................................................................ 88

References........................................................................................................................ 104

Chapter 4: Purification and crystal screening of two tRNA cleaving toxins.............................. 107

Abstract........................................................................................................................... 107

Introduction....................................................................................................................... 108

Materials and methods......................................................................................................... 109

Results and discussion......................................................................................................... 113

Conclusions/future directions................................................................................................. 116

Figures.............................................................................................................................. 117

References......................................................................................................................... 121

Chapter 5: Discussion........................................................................................................ 123

Abstract............................................................................................................................ 123

Understanding toxin superfamilies through structure................................................................... 123

Insights into RNA recognition by type III toxins......................................................................... 127

MazF-mt9 is a potential link between ToxN and MazF toxins......................................................... 130

Concluding remarks............................................................................................................... 132

Figures............................................................................................................................... 134

References.......................................................................................................................... 143

Appendix I: Uniformity of Peptide Release Is Maintained by

Methylation of Release....................................................................................................... 144

Summary............................................................................................................................. 145

Introduction........................................................................................................................ 145

Results............................................................................................................................... 148

Discussion........................................................................................................................... 155

Experimental procedures........................................................................................................ 157

Figures and tables................................................................................................................ 164

References.......................................................................................................................... 181

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