RNA and Protein Features Controlling Bacterial Translational Fidelity Open Access
Mattingly, Jacob (Fall 2024)
Abstract
Translation is the essential process by which all cells use information encoded in messenger RNA (mRNA) to direct the synthesis of proteins. To perform translation, cells use large complexes of RNA and protein known as ribosomes, which coordinate with transfer RNAs (tRNAs) and proteins known as translation factors to extend growing polypeptides through sequential addition of amino acids. The translational machinery has evolved mechanisms to protect the accuracy (fidelity) of protein synthesis, and studying these mechanisms can give us a clearer understanding of the processes underlying protein synthesis and assist in development of translation-targeting antibiotic drugs. To begin translation, ribosomes select a dedicated tRNA used only for initiation (called tRNAfMet in bacteria) and begin synthesizing proteins at a specific mRNA sequence (typically AUG, but sometimes GUG or UUG in bacteria). During initiation, tRNAfMet is recognized against all other cellular tRNAs through features including a series of three consecutive G-C base pairs in its anticodon stem, which interact with the ribosomal RNA (rRNA). Altering the sequence of the middle G-C base pair to C-G (yielding a mutant variant known as tRNAfMet M1) weakens interactions with the ribosome and appears to reduce the fidelity of initiation. The initiation factor IF2 restores normal initiation behavior to tRNAfMet M1, suggesting a previously unknown quality control role for IF2. Structural studies of initiation using tRNAfMet M1 demonstrate that IF2 strengthens the interaction of the tRNA with 16S rRNA nucleotide G1338, which may explain its ability to restore normal initiation in vitro. After initiation, ribosomes enter the elongation step of translation, where the growing protein is extended. Errors in elongation can be induced by aminoglycoside antibiotics, a critical class of ribosome-targeting antibiotics which are used in the treatment of severe or chronic infections that often respond poorly to other antibiotic classes. Aminoglycoside resistance via modification of their rRNA target threatens the efficacy of this class of drugs, although some aminoglycosides evade this mode of resistance better than others. Structural and simulation studies of the interaction of aminoglycosides with aminoglycoside-resistant ribosomes suggest several drug design principles that may be used to overcome resistance and preserve aminoglycoside efficacy.
Table of Contents
Abstract iv
Acknowledgements vi
Table of Contents iix
List of Figures x
List of Tables xiii
Chapter 1: Introduction 1
1.1. Architecture and function of translational machinery 1
1.2. Steps of the bacterial translation cycle 3
1.3. Maintenance and disruption of translational fidelity 5
1.4. Aminoglycoside antibiotics disrupt bacterial translational fidelity 7
1.5. Goals of this work 8
Chapter 2: Structural analysis of noncanonical translation initiation complexes 24
2.1. Abstract 25
2.2. Introduction 26
2.3. Results 29
2.4. Discussion 35
2.5. Experimental Procedures 39
2.6. Data Availability 44
2.7. Acknowledgements 45
2.8. References 52
2.9. Supplementary Data 55
Chapter 3: Basis for selective drug evasion of an aminoglycoside-resistance ribosomal RNA modification 78
3.1. Abstract 79
3.2. Introduction 79
3.3. Results 82
3.4. Discussion 99
3.5. Methods 102
3.6. Acknowledgements 109
3.7. Author contributions 109
3.8. Competing interests 109
3.9. References 110
3.10. Supplementary Information 122
Chapter 4: Discussion 141
4.1. Initiation is fine-tuned for a mixture of accuracy and efficiency 141
4.2. The role of IF2 in quality control during translation initiation 142
4.3. The role of G1338 in stabilizing tRNA conformations adjacent to the P site 145
4.4. tRNAfMet M1 does not cause frameshifting during initiation 146
4.5. Mechanisms of resistance evasion by 4,6-DOS aminoglycosides 149
4.6. Influence of drug structure flexibility 150
4.7. Influence of Ring I substituent identity 152
4.8. Influence of Ring II HABA group 152
4.9. Considerations for antibiotic design 153
4.10. Conclusions 155
4.11. References 160
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