Contact Us

Instructions

Frequently Asked Questions

ETD Help

Policies and Procedures

Copyright and Patents

Access Restrictions

Search ETDs:
Advanced Search
Browse by:
Browse ProQuest
Search ProQuest

Laney Graduate School

Rollins School of Public Health

Candler School of Theology

Emory College

Emory Libraries

New ETD website is now LIVE and located here: etd.library.emory.edu

Molecular mechanisms involved in maintaining ribosomal fidelity

Fagan, Crystal E. (2014)
Dissertation (173 pages)
Committee Chair / Thesis Adviser: Dunham, Christine
Committee Members: Corbett, Anita ; Cheng, Xiaodong ; Ortlund, Eric ; Reines, Daniel
Research Fields: Chemistry, Biochemistry
Keywords: Ribosome; Protein synthesis
Program: Laney Graduate School, Biological and Biomedical Sciences (Biochemistry, Cell & Developmental Biology)
Permanent url: http://pid.emory.edu/ark:/25593/gqwm3

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...15

Structural 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...35

Chapter 2: Reorganization of an intersubunit bridge induced by disparate 16S ribosomal ambiguity mutations mimics an EF-Tu-bound state...42

INTRODUCTION...44
RESULTS...46

Mutations 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...69

Chapter 3: Structural insights into translational recoding by frameshift suppressor tRNA SufJ...72

INTRODUCTION...74
RESULTS...76

Structural 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...102

Chapter 4: Molecular basis of ribosomal P-site quality control mechanism...105

INTRODUCTION...106
RESULTS...108

Near-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...126

How 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...141

APPENDIX 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...149

Structural studies of peptolide 12c bound to the 70S ribosome...149

CONCLUSION...150
METHODS...151
FIGURES...153
REFERENCES...159

Files

Access restricted until 2018-01-11

Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.