Transfer rna dynamics and transfer-messenger rna accommodation in bacterial ribosomes at the ...

161 Pages·2013·9.4 MB·English

by  May Daher Farhat

Wayne State University
DigitalCommons@WayneState
Wayne State University Dissertations
1-1-2012
Transfer rna dynamics and transfer-messenger rna
accommodation in bacterial ribosomes at the
single-molecule level
May Daher Farhat
Wayne State University,
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Recommended Citation
Farhat, May Daher, "Transfer rna dynamics and transfer-messenger rna accommodation in bacterial ribosomes at the single-molecule
level" (2012).Wayne State University Dissertations.Paper 565.
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TRANSFER RNA DYNAMICS AND TRANSFER-MESSENGER RNA
ACCOMMODATION IN BACTERIAL RIBOSOMES AT THE SINGLE-MOLECULE
LEVEL
by
MAY DAHER
DISSERTATION
Submitted to the Graduate School
of Wayne State University,
Detroit, Michigan
in partial fulfillment of the requirements
DOCTOR OF PHILOSOPHY
2012
MAJOR: CHEMISTRY (Biochemistry)
Approved by:

Advisor Date
DEDICATION

Dedicated to my family
ii
ACKNOWLEDGEMENTS

The accomplishment of this work would have been impossible without the support of
numerous people.
First and foremost, I would like to express my deep and sincere gratitude to my advisor
Dr. David Rueda for his guidance, support, and understanding. By working with him, I
have learned diverse ways to approach research problems that have enabled me to
grow as a researcher and be persistent on accomplishing my goals. I also would like to
thank my committee members, Dr.Christine Chow for her help, guidance especially
during my last year. I greatly appreciate her invaluable suggestions and constructive
comments to complete my dissertation. Dr. Philip Cunningham for his support and his
suggestions and critical comments. Dr. Claudio Verani for his suggestions and my
previous committee members, Dr. SantaLucia and Dr. Matthew Allen.
I am very thankful to all the past and current lab members. I really appreciate them for
being helpful. I am very grateful to Dr. Elvin Aléman for training me for the single-
molecule technique and other techniques in the lab. Dr. Amanda Solem for the useful
scientific discussion with her. Dr. Alfonso Brenlla for writing Matlab scripts for me and
proofreading my thesis. I would like to give very special thanks to Dr. Sharla Wood for
proofreading most of my professional documents and her help to improve my English
language as it is my second language. I am very thankful to Dr. Rajan Lammichhane,
Dr. Rui Zhao, Dr. Zhuojun Guo, Gayan, Bishnu, Chandani, Hansini, Imali, Pramodha,
Eric, Sanka and Todd Faner. I am very grateful to all the RNA club members for their
comments on my research and for allowing me to use their lab equipments whenever
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needed. I would like to thank Gayani from Dr. Chow lab for her help in my footprinting
and MALDI experiments, Dr. Tek Lamichhane for teaching me to purify ribosomes. Dr.
Chandrika Canugovi from Dr. Bhagwat lab for teaching me the expression and
purification of proteins. Shirin Fatme from Dr. Hendrickson lab for her help with my
phenylation assay.
Last but not least, I am very thankful to my husband, Bassem and my daughters
Rawan, Maryan and Ayah for their love, support, inspiration, and understanding. I would
never have achieved this much without them. I am also very thankful to my parents for
their encouragement in successful completion of my degree.
Thank you all.
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TABLE OF CONTENTS
Dedication.......................................................................................................................ii
Acknowelegments.........................................................................................................iii
List of Figures ..............................................................................................................xii
CHAPTER 1: Bacterial Ribosome Structure and Function ........................................1
1.1 Introduction of bacterial ribosome.....................................................................1
1.2 Structure and function of bacterial ribosomes..................................................2
1.2.1 Initiation of protein synthesis............................................................................5
1.2.2 Peptide elongation and translocation...............................................................6
1.2.3 Termination of translation and ribosome recycling...........................................8
1.3 Ribosome dynamics...........................................................................................10
1.3.1 Ribosome ratcheting mechanism...................................................................11
1.3.2 tRNA fluctuation inside ribosomes.................................................................12
1.3.3 L1 stalk motion during ribosomal ratcheting...................................................13
1.4 Ribosome rescue system..................................................................................13
1.4.1 Causes and effects of stalled ribosomes.......................................................13
1.4.2 Discovery, structure and function of transfer-messenger RNA (tmRNA).......16
1.4.3 SmpB is a cofactor of tmRNA........................................................................18
1.4.4 Trans-translation mechanism.........................................................................19
1.5 Dissertation summary........................................................................................22
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1.5.1 Aim 1. Determination of the SmpB-tRNA-like domain conformation in solution
and the effect of metal ions.....................................................................................23
1.5.2 Aim 2. Develop a single-molecule assay to monitor tmRNA accommodation
into a stalled ribosome............................................................................................23
1.5.3 Aim 3. Study of tmRNA entrance and accommodation in the ribosome at the
single-molecule level...............................................................................................24
CHAPTER 2: Methods..................................................................................................26
2.1 Fluorescence Spectroscopy..............................................................................26
2.2 Fluorescence resonance energy transfer........................................................28
2.3 Fluorescence anisotropy...................................................................................31
2.4 Steady-state FRET..............................................................................................33
2.5 Time-correlated single-photon counting (TCSPC)..........................................34
2.6 Single-molecule..................................................................................................35
2.6.1 Slide preparation for single-molecule experiments........................................37
2.6.2 Channel assembly..........................................................................................38
2.7 Single-molecule protein-induced fluorescence intensity...............................39
2.8 Oxygen scavenging system..............................................................................40
2.9 RNA-metal interaction........................................................................................41
2.10 Preparation of 70S ribosomes and mRNAs...................................................42
2.11 Fluorophore labeling........................................................................................44
CHAPTER 3: Fluorescence Characterization of the tRNA-like Domain of tmRNA in
Complex with SmpB (61).............................................................................................46
3.1 Introduction to tmRNA-SmpB complex structure and function.....................46
vi
3.2 Materials and methods.......................................................................................48
3.2.1 RNA purification and fluorophore labeling......................................................48
3.2.2 SmpB expression and purification..................................................................49
3.2.3 Application of anisotropy in our system..........................................................50
3.2.3 Quantum yield calculation..............................................................................51
3.2.5 Steady State FRET applications....................................................................53
3.2.6 Time-resolved FRET (trFRET) measurements..............................................53
3.3 Results.................................................................................................................55
3.3.1 SmpB binds tmRNAΔ tightly and specifically.................................................55
3.3.2 Mg2+ but not Na+ ions inhibit SmpB binding...................................................58
3.3.3 SmpB does not induce global conformational changes in tmRNA∆...............60
3.3.4 Mg2+ ions compress the tmRNA∆ structure. ..................................................64
3.4 Conclusions........................................................................................................67
CHAPTER 4: tRNAs Dynamics in Ribosomes...........................................................71
4.1 Introduction.........................................................................................................71
4.2 Materials and Methods......................................................................................72
4.2.1 Preparation of 70S ribosomes and mRNAs...................................................72
4.2.2 Expression and purification of EF-Tu and EF-G............................................73
4.2.3 GTPase activity assay in the presence of 70S ribosome...............................74
4.2.4 tRNAs fluorophore labeling............................................................................75
4.2.5 Phenylalanyl tRNA synthetase (pheRS) expression and purification.............77
4.2.6 Characterization of tRNA species..................................................................79
4.2.7 Ternary complex formation............................................................................83
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4.2.8 Ribosome complexes.....................................................................................84
4.2.9 Antibiotics assays...........................................................................................84
4.2.10 Single-molecule PIFE measurements..........................................................85
4.3 Results.................................................................................................................87
4.3.1 Fluorescence is only observed when all the components are present...........87
4.3.2 Sm-PIFE reveals two populations distribution of the PRE complex inside
ribosomes................................................................................................................87
4.3.3 EF-G in the presence of non-hydrolysable GTP favors the static population
inside the ribosome.................................................................................................90
4.3.3 Mg2+ alters the dynamics of tRNA in the ribosome........................................91
4.3.6 Puromycin binding increases the fraction of static molecules........................92
4.3.5 Viomycin shifts the dynamic of tRNA toward static motion............................93
4.3.6 Paromomycin binding shifts the dynamic equilibrium of tRNA toward
multistate motion.....................................................................................................94
4.3.7 Characterization of binding of deacylated tRNA to the ribosome...................95
4.4 Conclusions........................................................................................................95
4.4.1 Spontaneous fluctuation of PRE translocation complex is a multistep
processes................................................................................................................95
4.4.2 Mg2+ may stabilize tRNA-ribosome interactions.............................................97
4.4.3 Evidence of the relation between ribosome ratcheting and fluorescence
fluctuation................................................................................................................97
CHAPTER 5: Monitoring the Entrance of the tmRNA-SmpB Complex into Stalled
Ribosomes Using Sm-FRET......................................................................................100
viii
5.1 Introduction.......................................................................................................100
5.2 Materials and methods.....................................................................................102
5.2.1 In vivo and in vitro transcription of tmRNA...................................................102
5.2.2 SmpB mutation............................................................................................105
5.2.3 Labeling of SmpB by Cy5 acceptor fluorophore...........................................107
5.2.4 Alanyl-tRNA synthetase (AlaRS) expression and purification......................108
5.2.5 The quaternary complex formation..............................................................110
5.2.6 Dipeptide reaction........................................................................................110
5.2.7 Single-molecule experiment.........................................................................111
5.3 Results...............................................................................................................111
5.3.1 Transfer-messenger RNA entrance into the stalled ribosome is confirmed by a
thin layer chromatography TLC experiment..........................................................111
5.3.2 Sm-FRET analysis of the tmRNA delivery to the stalled ribosomes............112
5.3.2 Tetracycline blocks progression of tmRNA into accommodation.................114
5.3.4 Kirromycin interferes with the full accommodation of tmRNA in the A site..116
5.3.5 The presence of non-hydrolysable GTP (GDPNP) prevents the tmRNA
progression into the full accommodation state......................................................116
5.3.6 Effect of the length of the reading frame of mRNA on the tmRNA
accommodation.....................................................................................................117
5.3.7 SmpB binds the ribosome in the absence of tmRNA...................................118
5.4 Conclusions......................................................................................................120
CHAPTER 6: Conclusions and Future Directions...................................................122
References..................................................................................................................128
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