Molecular mechanisms involved in maintaining ribosomal fidelity Open Access
Fagan, Crystal E. (2014)
Abstract
The ability to faithfully translate the genetic instructions into functional proteins is critical. The process of translation is carried out by the ribosome using a set of finely tuned tRNA adapter molecules to translate the messenger RNA three nucleotides at a time. In order to accurately decode the mRNA, the ribosome must both select the correct tRNA to decode the mRNA and preserve the three nucleotide mRNA reading frame. If errors do occur, a quality control mechanism is activated to terminate the synthesis of that erroneous protein. The work presented here analyzes the molecular mechanisms responsible for maintaining intrinsic ribosomal fidelity in bacteria. In order to understand how correct tRNA decoding is signaled across the ribosome, I structurally characterized 16S rRNA ribosomal ambiguity (ram) mutations that resulted in a strong miscoding phenotype. Through this work I have identified a global regulator of translational fidelity, the intersubunit bridge B8. This bridge increases the stringency of mRNA decoding by negatively regulating GTPase activation, an essential process in tRNA selection. To understand the role of tRNA structure in maintaining the mRNA reading frame, I characterized a tRNA that contains an expanded anticodon stem-loop. Our results show distortions in the tRNA shape could impede gripping interactions with the ribosome and other translation factors resulting in incorrect mRNA decoding. Finally, I investigated the molecular mechanisms responsible for the identification of errors after they have been incorporated into the nascent peptide chain. This work suggests incorrect tRNA-mRNA interaction could alter the position of the mRNA. These different research projects have helped to provide a more complete understanding of the intrinsic ribosomal mechanisms responsible for maintaining translational fidelity
Table of Contents
Chapter 1. General Introduction...1 The bacterial ribosome...4 The tRNA structure...6 The translation cycle...8 Translation initiation...8 Translation elongation...9 Translation termination...12 Ribosome recycling...12
The role of the ribosome in translational fidelity...12
The kinetic proofreading model...15Structural basis of cognate tRNA recognition...16
An integrated model for tRNA selection...18
Loss of translational fidelity through mRNA frameshifts...19
Retrospective editing...21 Summary...22 FIGURES...24 REFERENCES...35Chapter 2: Reorganization of an intersubunit bridge induced by disparate 16S ribosomal ambiguity mutations mimics an EF-Tu-bound state...42
INTRODUCTION...44 RESULTS...46Mutations G299A and G347U promote activation of EF-Tu...46
Crystallization and structural analysis of T. thermophilus G299A and G347U ribosomes...47
G347U directly alters h14, thereby disrupting bridge B8...48
G299A indirectly alters h14, also disrupting bridge B8...49
Comparison of G347U and G299A structures with previous 70S structures...50
DISCUSSION...51 MATERIALS AND METHODS...54 ACKNOWLEDGEMENTS...56 FIGURES...57 REFERENCES...69Chapter 3: Structural insights into translational recoding by frameshift suppressor tRNA SufJ...72
INTRODUCTION...74 RESULTS...76Structural determination of ASLSufJ bound to +1 suppressible codons in the 70S A site...78
The C31.5 insertion in ASLSufJ results in conformational rearrangements in the stem of the ASL...80
Anticodon stem register is maintained in ASLSufJ despite alteration of the conserved 32-38 base pair...80
Projection of tRNASufJ in the A and P sites indicates potential rearrangements due to steric clashes...81
DISCUSSION...82 FIGURES...90 REFERENCES...102Chapter 4: Molecular basis of ribosomal P-site quality control mechanism...105
INTRODUCTION...106 RESULTS...108Near-cognate tRNA in the P site causes a change in the mRNA reading frame...108
Elongation of the mRNA message allows for the correct positioning of the P-site codon...109
The U•U mismatch in the first position is stabilized by the t6A37 modification and alters the 3' path of the mRNA...110
The U•U mismatch in the second position narrows the codon-anticodon helix...111
Movement of the third position U•U mismatch destabilizes the mRNA kink...111
CONCLUSIONS/FUTURE STUDIES...112 MATERIALS AND METHODS...113 FIGURES...114 REFERENCES...125 CHAPTER 5. General Discussion...126How is selection of the correct tRNA signaled?...126
Is the intersubunit bridge B8 a global regulator of the GTPase activation?...129
How is the three-nucleotide mRNA reading frame maintained?...129
How is the low efficiency of frameshift suppression by tRNASufJ maintained?...132
How does a near-cognate tRNA in the P site alter ribosomal fidelity?...134
CONCLUSION...135 FIGURES...137 REFERENCES...141APPENDIX 1: Macrolide-Peptide Conjugates as Probes of the Path of Travel of the Nascent Peptides through the ribosome...144
ABSTRACT...145 INTRODUCTION...146 RESULTS AND DISCUSSION...149Structural studies of peptolide 12c bound to the 70S ribosome...149
CONCLUSION...150 METHODS...151 FIGURES...153 REFERENCES...159
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