Structure and Function of Methyltransferases that Modify the Ribosome and Ribosome-Associated Factors 公开
Kuiper, Emily Gretchen (2016)
Published
Abstract
The ribosome is the molecular machine responsible for translating messenger RNA into proteins. Ribosome assembly and function is modulated by posttranscriptional and posttranslational modifications of the ribosomal components and its associated factors. Additionally, other modifications allow the ribosome to adapt to stressors in the environment. While many ribosomal modifications have been identified, less is known about the structural and molecular mechanisms of catalysis of the modification enzymes. In this dissertation I elucidate novel structural and functional characteristics of three methyltransferases that aid bacteria in adapting to environmental stressors such as infection of a host or survival in the presence of antibiotics. Deletion of EftM, the EF-Tu trimethyltransferase, decreases Pseudomonas aeruginosa adherence to and infection of host cells. I show via homology modeling and mutation of the putative S-adenosyl-L-methionine binding motif that EftM belongs to Class I methyltransferases. Further, I show that the observed temperature regulation of the modification is due to a novel regulatory mechanism where the methyltransferase unfolds at the restrictive temperature, resulting in the observed repression of EF-Tu methylation. Loss of the Mycobacterium tuberculosis constitutive ribosomal RNA methyltransferase TlyA renders ribosomes resistant to the antibiotic capreomycin. I identified and characterized a novel auxiliary cosubstrate-binding motif, within an interdomain linker that is essential for cosubstrate binding. This motif is conserved in TlyA orthologs, suggesting that it is functionally important. We speculate that this motif likely coordinates substrate recognition via an amino-terminal domain with cosubstrate binding and catalysis. The thiostrepton-resistance methyltransferase (Tsr) confers antibiotic resistance in the thiostrepton antibiotic-producing bacterium Streptomyces azureus. Here, I elucidate Tsr's cosubstrate binding affinity and catalytic mechanism and show that as a dimer, each protomer acts independently to bind cosubstrate and methylate its 23S rRNA substrate. Furthermore, I present a novel substrate recognition mechanism where Tsr induces conformational changes in the rRNA substrate prior to catalysis. My studies begin to elucidate the coordination involved between the two protomers and between substrate-recognition and catalytic domains for proper enzyme function. Together, my studies describe the molecular mechanisms of methyltransferases which complements cellular studies that examine the function of a modification in a bacterium or in adaptation to stress.
Table of Contents
1 CHAPTER 1: INTRODUCTION TO DISSERTATION 1
1.1 The Ribosome and the Translational Machinery 1
1.1.1 Translation Initiation 2
1.1.2 Translation Elongation 3
1.1.3 Translation Termination 4
1.2 Antibiotics Inhibit Important Functional Areas of the Ribosome 5
1.2.1 Peptide Exit Tunnel 5
1.2.2 Peptidyl-Transferase Center 6
1.2.3 30S Subunit and Decoding Center 6
1.2.4 GTPase Activation Center and GTPase 7
1.3 Methylation of the ribosomal machinery: Fine tuning ribosome function, antibiotic resistance and pathogenesis 8
1.3.1 Posttranscriptional Modifications of rRNA 9
1.3.1.1 Constitutive modifications aid in ribosome function:30S subunit 9
1.3.1.2 Constitutive modifications aid in ribosome function:50S subuniT 10
1.3.1.3 Constitutive modifications aid in antibiotic binding and inhibition 11
1.3.1.4 Posttranscriptional modifications can confer antibiotic resistance 12
1.3.2 Posttranslational Modifications 14
1.3.2.1 Ribosomal protein modifications 14
1.3.2.2 Modifications of ribosome factors 15
1.3.2.3 Control of EF-Tu function by modification during different phases of bacterial growth 15
1.4 Prevailing Questions in methyltransferase structure and function 16
1.5 References 29
2 CHAPTER 2: PSEUDOMONAS AERUGINOSA EFTM IS A THERMOREGULATED METHYLTRANSFERASE 45
2.1 Abstract 46
2.2 Introduction 46
2.3 Experimental Procedures 49
2.3.1 Bacterial strains, plasmids and primers 49
2.3.2 Plasmid construction 49
2.3.3 Protein expression and purification 50
2.3.4 Preparation of P. aeruginosa whole-cell extracts 51
2.3.5 Immunoblot analysis 51
2.3.6 EftM homology modeling 51
2.3.7 In vitro methyltransferase assay 52
2.3.8 MS analysis 52
2.3.9 Isothermal titration calorimetry 54
2.3.10 Circular dichroism spectroscopy 54
2.3.11 Differential scanning fluorimetry 55
2.4 RESULTS 552.4.1 EftM is a SAM-dependent methyltransferase 55
2.4.2 EftM is necessary and sufficient to methylate EF-Tu 57
2.4.3 EF-Tu methylation by EftM is distributive 58
2.4.4 EftM methyltransferase activity is thermosensitive 59
2.4.5 Structural thermolability regulates EftM activity 59
2.5 DISCUSSION 60
2.6 ACKNOWLEDGEMENTS 63
2.7 REFERENCES 74
3 Chapter 3: A novel motif required for S-adenosyl-L-methionine cosubstrate binding by the 2´-O-methyltransferase Tlya from Mycobacterium tuberculosis 79
3.1 SUMMARY 80
3.2 INTRODUCTION 80
3.3 RESULTS 82
3.3.1 Construct Design, Protein Expression and Purification of TlyA for Structural Studies 82
3.3.2 The TlyA CTD Adopts a Class I Methyltransferase Fold 84
3.3.3 The Isolated TlyA CTD Protein Does Not Bind SAM 85
3.3.4 The RAWV Tetrapeptide Interdomain Linker is Critical for TlyA CTD-SAM Interaction 86
3.3.5 Trp62 and Val63 are Critical for SAM Binding 88
3.3.6 Structural Plasticity of RAWV Motif 89
3.4 DISCUSSION 92
3.5 EXPERIMENTAL PROCEDURES 94
3.5.1 TlyA Construct Design and Site-directed Mutagenesis 94
3.5.2 TlyA Protein Expression and Purification 95
3.5.3 CD Spectroscopy 96
3.5.4 RT Analysis of 16S and 23S rRNA Methylation 96
3.5.5 Protein Crystallization and Structure Determination 97
3.5.6 Isothermal Titration Calorimetry Analysis of SAM and SAH Binding 98
3.5.7 Analysis of TlyA Domain Association by Gel Filtration Chromatography983.5.8 SAM and NTD Modeling 99
3.6 ACCESSION NUMBERS 99
3.7 ACKNOWLEDGEMENTS 99
3.8 SUPPLEMENTAL INFORMATION 112
3.9 REFERENCES 115
4 Chapter 4: Binding induced RNA conformational changes control substrate recognition and catalysis by the thiostrepton-resistance methyltransferase (Tsr) 120
4.1 SUMMARY 121
4.2 INTRODUCTION 121
4.3 EXPERIMENTAL PROCEDURES 124
4.3.1 Tsr purification and mutagenesis 124
4.3.2 RNA in vitro transcription 124
4.3.3 Hydroxyl radical and ribonuclease RNA structure probing 125
4.3.4 RNA UV Melting 126
4.3.5 Fluorescence polarization 126
4.3.6 Methylation Assays 126
4.3.7 Electrophoretic mobility shift assay 127
4.3.8 Partial Proteolysis 127
4.4 RESULTS 127
4.4.1 The Tsr NTDs aid in rRNA binding and are necessary for catalysis 127
4.4.2 Identification of the RNA binding surface and binding-induced perturbations in the RNA structure by Tsr 130
4.4.3 RNA conformational changes induced by Tsr are dependent on its NTD 132
4.4.4 Stabilizing the 58 nt RNA tertiary structure does not interfere with RNA conformational changes induced by RNase V1 134
4.4.5 Tsr mutants R162A and R26A discriminate between RNA binding and induced conformational changes necessary for catalysis 135
4.5 DISCUSSION 137
4.6 ACKNOWLEDGEMENTS 140
4.7 REFERENCES 152
5 Chapter 5: Functional rolEs in S-Adenosyl-L-Methionine binding and catalysis for active site residues of the thiostrepton-resistance methyltransferase 157
5.1 ABSTRACT 158
5.2 INTRODUCTION 158
5.3 MATERIALS AND METHODS 160
5.3.1 Protein expression & purification 160
5.3.2 Size exclusion chromatography 161
5.3.3 Methylation assays 161
5.3.4 Isothermal titration calorimetry 162
5.3.5 Circular dichroism spectroscopy 163
5.4 RESULTS 163 5.4.1Characterization of SAM binding and turnover by Tsr 163 5.4.2Effect of active site mutations on Tsr activity 165
5.4.3 Thermodynamics of SAM binding and kinetics of SAM turnover by select Tsr mutants 166
5.4.4 N129 is involved in the structural organization of Tsr 167
5.5 DISCUSSION 168
5.6 ACKNOWLEDGEMENTS 170
5.7 SUPPLEMENTARY MATERIAL 177
5.8 REFERENCES 183
6 Chapter 6: DISCUSSION 187
6.1 STRUCTURAL INSIGHTS 187
6.1.1 Structural Instability Regulates EftM Activity 187
6.1.2 TlyA Linker May Coordinate Interdomain Interactions 188
6.1.3 Tsr Protomers Act Independently of Each Other in Catalysis 189
6.2 COSUBSTRATE BINDING 190
6.2.1 Identification and Characterization of the SAM Binding Motif in EftM 190
6.2.2 A Novel Auxiliary SAM Binding Motif in TlyA 190
6.2.3 Tsr Cosubstrate Recognition and Catalysis 191
6.3 SUBSTRATE RECOGNITION 191
6.3.1 A New Model of Tsr Substrate Recognition 192
6.3.2 TlyA Recognizes Two Different Substrates 193
6.3.3 EF-Tu Lysine Recognition by EftM 194
6.4 MODIFICATION ENZYMES AID IN BACTERIAL ADAPTATION 195
6.4.1 TlyA is a Methyltransferase and Hemolysin 196
6.4.2 EftM Mediated Trimethylation aids in P. aeruginosa Infection 197
6.4.3 Thiopeptides and the Antibiotic Resistome 197
6.5 FUTURE DIRECTIONS 198
6.5.1 EftM 198
6.5.2 TlyA 199
6.5.3 Tsr 199
6.6 CONCLUDING REMARKS 200
6.7 REFERENCES 202
About this Dissertation
| School | |
|---|---|
| Department | |
| Subfield / Discipline | |
| Degree | |
| Submission | |
| Language |
|
| Research Field | |
| 关键词 | |
| Committee Chair / Thesis Advisor | |
| Committee Members |
Primary PDF
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
Structure and Function of Methyltransferases that Modify the Ribosome and Ribosome-Associated Factors () | 2018-08-28 15:27:40 -0400 |
|
Supplemental Files
| Thumbnail | Title | Date Uploaded | Actions |
|---|