Investigating the regulation of bacterial RNases and their impact on gene expression Restricted; Files Only
Tanquary, Julia (Summer 2025)
Abstract
While RNases are typically discussed in the context of RNA turnover, bacterial RNases play important roles in gene regulation and responding to the environment. Bacterial RNases allow cells to quickly respond to environmental stressors such as phage infection, by selectively cleaving phage RNA. RNases also act indirectly by cleaving transcripts to suppress gene expression. Since RNases play such important roles in cellular processes, it is vital that their activity is tightly regulated. The work presented in this dissertation investigates how specific bacterial RNases are regulated and identifies the consequences of misregulation. We structurally and biochemically characterize an RNase from methicillin-resistant Staphylococcus aureus, YhaM, and identify that YhaM activity is regulated by incorporation of different YhaM protomers in the functional YhaM complex. Furthermore, YhaM in the correct composition of protomers is required for virulence. Additionally, we define the mechanism of regulation of an Escherichia coli RNase that is a component of a toxin-antitoxin system, RelB-RelE. We find that the suppression of the RelE toxin is distinct from that of a structurally similar toxin-antitoxin complex. This difference in toxin suppression could account for the ability of each toxin-antitoxin complex to bind and repress its own operator. Expanding on our toxin-antitoxin research, we investigated the different effects of overexpression of a toxin that is an RNase and compared effects toxin overexpression to the effects of endogenous toxin activation. Toxin overexpression results in more mRNA cleavage sites than endogenous toxin activation. These results challenge the current method of studying toxin targets and the physiological effects of toxin activation. The research presented here defines different forms of RNase regulation and has implications for RNase specificity. This work expands our understanding of important bacterial processes and the role that RNases play in regulating gene expression.
Table of Contents
Chapter 1 1
Introduction 1
1.1 Gene Expression and the adaptability of bacterial growth 1
1.2 RNases and their role in regulating gene expression 2
1.3 YhaM, an RNase the indirectly regulated global translation and contributes to virulence 4
1.4 Toxin-antitoxin systems and the direct regulation of translation 6
1.5 Introduction to dissertation research 12
1.6 Figures 15
1.7 References 23
Chapter 2 33
An unusual structural assembly of Staphylococcus aureus exoribonuclease YhaM essential for RNA processing and virulence 33
2.1 Abstract 34
2.2 Significance 35
2.4 Introduction 35
2.5 Results 37
2.6 Discussion 44
2.7 Materials and Methods 47
2.8 Acknowledgements 56
2.9 Figures 58
2.10 Tables 76
2.11 References 80
Chapter 3 88
The Escherichia coli RelB antitoxin C-terminus is essential for RelE toxin suppression and transcriptional repression 88
3.1 Abstract 89
3.2 Introduction 90
3.3 Results 91
3.4 Discussion 99
3.5 Experimental Procedures 102
3.6 Acknowledgements 111
3.7 Figures 113
3.8 Tables 129
3.9 References 132
Chapter 4 140
Activation of chromosomally-encoded YafQ via inducible degron: the phenotypic and transcriptomic effects 140
4.1 Abstract 141
4.2 Introduction 142
4.3 Results 145
4.4 Discussion 149
4.5 Limitations of study 151
4.6 Materials and Methods 152
4.7 Figures 158
4.8 Tables 169
4.9 References 170
Chapter 5 177
5.1 Overview of Bacterial RNases and their roles in regulating cellular processes 177
5.2 YhaM – unique structural requirements for virulence 178
5.3 Regulation of toxin activation varies between structurally similar TA systems 180
5.4 Endogenous toxin activation results in different RNA cleavage than toxin overexpression 184
5.5 Technical applications of dissertation findings 187
5.6 Conclusions and Future Implications 189
5.7 Figures 191
5.8 References 195
About this Dissertation
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File download under embargo until 21 August 2027 | 2025-07-06 17:04:32 -0400 | File download under embargo until 21 August 2027 |
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