Structural and functional studies of a toxin-anti toxin system involved in translational inhibition Open Access

Schureck, Marc (2016)

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Bacteria regulate protein synthesis during environmental stress as a survival mechanism. One way translation is regulated is through cleavage of ribosome-bound mRNA by ribosome-dependent toxins. This mRNA cleavage stops the synthesis of the protein encoded by the cleaved-mRNA, conserves nutrients and likely plays an important, yet unknown, role in altering the spectrum of proteins translated during stress. A very unique feature of ribosome-dependent toxins is that they can recognize and cleave several mRNA sequences on the ribosome. In this dissertation, the molecular mechanism of recognition and cleavage of adenosine-rich mRNA codons by the Proteus vulgaris HigB toxin, which was originally identified on a drug-resistance plasmid from a P. vulgaris urinary tract infection, was investigated. Structural and biochemical studies reveal that the HigB toxin displays degenerate substrate specificity by creating two A-site nucleotide-binding pockets capable of interacting with numerous nucleotides. Surprisingly, the third nucleotide-binding pocket of HigB is adenosine-specific. Recognition of the third A-site nucleotide appears to be a distinct feature of ribosome-dependent toxins and likely influences which mRNAs are targeted for cleavage during environmental stress. Ribosome-dependent toxins must be highly regulated. The HigA antitoxin binds to and inactivates the HigB toxin when cells are not in a stressed state. Structural investigation shows that HigA and HigB form a tetrameric complex consisting of two HigA proteins and two HigB proteins. This structure reveals that HigA does not inactivate HigB through direct interactions with the HigB active site, as observed in many other toxin-antitoxin complexes. Instead, HigA binding to HigB likely inhibits HigB by blocking association of HigB with the ribosome. The knowledge of how HigB activity is regulated and its unique specificity provides a molecular framework for scientists to uncover how ribosome-dependent toxins control translation during environmental stress.

Table of Contents

Chapter 1. 1

Introduction: Toxin-antitoxin systems. 1

1.1 Abstract. 1

1.2 Antibiotics. 2

1.3 Molecular mechanisms of antibiotic action. 3

1.4 Antibiotics and the ribosome. 4

1.5 Bacteria that are antibiotic tolerant - Persisters. 6

1.6 Mechanisms of persister cell formation. 7

1.7 The bacterial stringent response. 8

1.8 Activation of the stringent response through the lack of amino acids. 8

1.9 Toxin-Antitoxin Systems. 9

1.10 Transcriptional regulation of toxin-antitoxin systems. 11

1.11 Molecular targets of toxin proteins. 13

1.12 Toxin-antitoxin systems and persister cell formation. 13

1.13 Other roles of toxin-antitoxin systems. 15

1.14 Translational inhibition during stress. 16

1.15 Ribosome-dependent toxins. 16

1.16 The HigB-HigA toxin-antitoxin system. 18

1.17 Questions addressed. 19

1.18 References. 30

Chapter 2. 41

Structure of the P. vulgaris HigB-(HigA)2-HigB toxin-antitoxin complex. 41

2.1 Abstract. 42

2.2 Introduction. 42

2.3 Experimental procedures. 44

2.3a HigBA expression and purification. 45

2.3b Crystallization, X-ray data collection and structural determination of HigBA complexes. 46

2.3c Size exclusion chromatography (SEC) assays. 47

2.3d Electrophoretic mobility shift assay (EMSA). 48

2.3e Molecular modeling HigB on the 70S ribosome. 48

2.4 Results. 49

2.4a Structural determination of the HigB-(HigA)2-HigB complex. 49

2.4b HigB adopts a microbial RNase fold. 50

2.4c The interface between HigA and HigB is novel. 51

2.4d HigA monomer contains an intact DNA binding domain. 52

2.4e HigA mediates the formation of the HigB-(HigA)2-HigB complex. 53

2.4f HigA does not mask the HigB active site. 54

2.4g A HigB-(HigA)2-HigB tetramer is required to interact with its DNA operator. 54

2.5 Discussion. 56

2.6 Acknowledgments. 60

2.7 Footnotes. 60

2.8 References. 78

Chapter 3. 89

mRNA bound to the 30S subunit is a HigB endonuclease substrate. 89

3.1 Abstract. 90

3.2 Introduction. 91

3.3 Results. 93

3.3a HigB toxin can target the initiation step of translation. 93

3.3b Structural basis of HigB toxin recognition of the 30S subunit. 94

3.4 Discussion. 99

3.5 Methods and materials. 103

3.5a Strains and plasmids. 103

3.5b Purification of E. coli 30S ribosomes. 103

3.5c HigB expression and purification. 104

3.5d mRNA cleavage assays. 105

3.5e Structural determination of the 30S-HigB complex. 105

3.5f Bacterial growth assays. 106

3.6 Acknowledgements. 107

3.7 References. 116

Chapter 4. 123

Defining the mRNA recognition signature of a bacterial toxin protein. 123

4.1 Abstract. 125

4.2 Significance. 125

4.3 Introduction. 126

4.4 Results & Discussion. 128

4.4a Structural determination of HigB-ribosome complexes. 128

4.4b Recognition of the A site by HigB involves distortion of the mRNA. 129

4.4c A-site nucleotide requirements for HigB cleavage. 131

4.4d Cross talk between A-site nucleotides drives efficient HigB recognition of mRNA. 134

4.4e A single HigB residue modulates codon selectivity. 135

4.5 Conclusions. 136

4.6 Materials & Methods. 139

4.6a Strains and plasmids. 139

4.6b Sequence and structural alignments. 139

4.6c Wild-type HigB, HigB ΔH92 and HigB N71A expression and purification. 140

4.6d Structural determination of HigB. 140

4.6e Structure Determination of 70S-HigB complexes. 141

4.6f mRNA cleavage assays. 142

4.6g Remodeling of the 70S-YoeB mRNA A-site mRNA. 143

4.7 Acknowledgements. 143

4.8 References. 167

Chapter 5. 175

Mechanism of endonuclease cleavage by the HigB toxin. 175

5.1 Abstract. 176

5.2 Introduction. 176

5.3 Materials and methods. 179

5.3a Strains and plasmids. 179

5.3b 70S purification, complex formation and structure determination of the 70S-HigB

precleavage state complex. 179

5.3c Bacterial growth or toxicity assays. 180

5.3d Single-turnover kinetic measurements. 181

5.3e Structure determination of HigB variants. 182

5.4 Results. 183

5.4a Structure determination of the 70S - wild-type HigB complex. 183

5.4b Effect of HigB variants on growth suppression. 184

5.4c HigB residues His54, Asp90, Tyr91 and His92 are critical for mRNA cleavage. 186

5.4d His92 is critical for optimal organization of the HigB active site. 187

5.5 Discussion. 187

5.6 Acknowledgment. 190

5.7 Funding. 190

Chapter 6. 215

Conclusion. 215

6.1 Abstract. 215

6.2 Introduction. 216

6.3 Toxin activity can have varying effects on translation. 217

6.4 Potential ways in which toxins reshape the translational landscape. 219

6.5 Towards an understanding of the roles of ribosome-dependent toxins. 221

6.6 Molecular studies will aid the accurate annotation of ribosome-dependent toxins. 222

6.7 Mechanism of antitoxin inhibition of toxin function. 224

6.8 Molecular mechanisms of cleavage of mRNA by ribosome-dependent toxins. 230

6.9 Concluding remarks. 235

6.10 References. 249

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