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Proquest Dissertations University of Alberta Investigating the mechanism of FinO-mediated inhibition of bacterial conjugation by David Cameron Arthur A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biochemistry Edmonton, Alberta Spring 2009 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington OttawaONK1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Vote reference ISBN: 978-0-494-55313-8 Our file Notre reference ISBN: 978-0-494-55313-8 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Nnternet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. •+• Canada To: Tom and Fiona Abstract Bacterial conjugation is repressed by a two-component system comprising the antisense RNA FinP and its protein co-factor FinO. FinO protects FinP from cellular degradation and mediates base-pairing to its target traJ mRNA leading to translational inhibition of the transcriptional activator TraJ and subsequent downregulation of conjugation genes. In this work, we explore the mechanism of FinO-mediated intermolecular duplexing of FinP and traJ mRNA. Biochemical assays show that FinO performs strand exchange on an SLII-derived duplex RNA and mediates duplexing of SLII to its complement SLIIc from traJ mRNA in an ATP- independent fashion reminiscent of RNA chaperones. This strongly suggests that FinO destabilizes base pairs within SLII and SLIIc which present a barrier to duplex formation. Also, the strand exchange and duplexing processes are shown to be important for repression of bacterial conjugation in vivo. Scanning alanine mutagenesis shows that the strand exchange area of FinO resides in a lysine-rich area on the N-terminal a-helix containing a solvent exposed tryptophan residue which is hypothesized to play a role in destabilizing portions of the stem regions of SLII and SLIIc. N-terminal truncation mutants abolishing strand exchange and duplexing bind significantly tighter than wild-type FinO indicating that FinO uses a portion of its binding energy to destabilize RNA duplexes. Secondly, we examine the details of how FinO binds to its target RNAs. We attempt to crystallize a FinO-SLII complex using a variety of RNA and protein constructs. However, despite our efforts, the best crystals only diffract to 4.5 A. Site specific protein-RNA cross-linking experiments highlight positively charged regions of the N-terminal alpha-helix and globular region of FinO which contact SLII. RNase footprinting studies locate the areas of SLII contacting FinO. FinO protects the lower 3' side of the SLII stem and 3' tail from RNase digestion. The loop region of SLII is not contacted by FinO as shown previously. N-terminal truncation mutants exhibit similar RNase footprinting patterns as FinO, indicating the globular region of FinO plays the primary role in binding to SLII. A model is proposed highlighting the relationship between the binding and catalytic components of FinO-mediated inhibition of conjugation. Acknowledgements First, I would like to thank my supervisor, Dr. Mark Glover, for his guidance and patience throughout my Ph.D. program. Thank you for taking a chance on a student who did not have the best academic record but was eager to jump into the research environment. Thanks for marching me up Heart mountain, Roche Miette, Mt. Glasgow (almost...), and Mt. Rundle. Finally, thanks for the chair lift conversations at the Colorado "Ski-stone" meetings. I would need another thesis to fully thank my FinO colleague and good friend Dr. Ross Edwards. Ross patiently taught me almost everything I know today and more importantly shared many experiences with me, good and bad. I think of Ross when I hear the words: All Blacks, Wolfenstein, Black Dog, baked glassware, Linux, Friday morning hockey, and underscore. Dr. Steven Chaulk is the Newfoundlander who taught me everything I know about RNA. Since this makes up a sizeable chunk of this thesis, I owe him a great deal of thanks. I would also like to thank Steve for being such a good friend. Sitting on his front porch sharing a cold one, "jamming", torturing ourselves watching the Leafs, and winter BBQs were just a few things I'll remember. I would like to thank Dr. Alex Ghetu for helping me get started with the FinO project. Alex taught me a lot of techniques and helped out with many experiments in this thesis. I would like to thank the other members of the Glover Lab, past and present, for their helpful discussions and friendship: Scott, Jason, Nina, Megan, Gina, Jun, Stephen, Charles, Danny, Ruth, Joyce, Jody, and Diana. Thank you to my collaborators and other departmental members for helpful discussions during my graduate studies: Dr. Laura Frost, Dr. Michael Gubbins, Dr. Leo Sypracopoulos, Dr. Andrew MacMillan, Dr. Paul Scott, Dr. Tracy Raivio, Dr. Daelynn Buelow, Craig Garen and Sheraz Khan. Finally, thank you to my fellow graduate students from the Department of Biochemistry for so many "good times". Table of Contents Page Chapter 1: Introduction Bacterial conjugation 1 Repression of conjugation - from past to present 4 Themes in antisense RNA regulation of gene expression in prokaryotes 15 RNA chaperones 21 Organization of the thesis 28 References 39 Chapter 2: FinO acts as an RNA chaperone to facilitate F'inP-traJ mRNA interactions Overview 49 Introduction 50 Results 51 Discussion 59 Materials and Methods 63 References 80 Chapter 3: Strategies for crystallization of the FinO-SLII RNA complex Overview 85 Introduction 85 Results 89 Discussion and future directions 97 Materials and Methods 100 References 120 Chapter 4: Examining the molecular details of the interaction of FinO with its target RNAs Overview 125 Introduction 125 Results 128 Discussion and future directions 136 Materials and Methods 141 References 157 Chapter 5: General Discussion Overall summary 161 Orientation of FinO on FinP SLII 163 Implications for FinP and traJ mRNA duplexing 165 The N-terminus of FinO and disorder as a possible mechanism for its chaperoning function 170 Concluding remarks: The bigger picture 171 References 177 Appendix A: Assaying FinO structural homologs for RNA chaperone activity Introduction 181 Results and discussion Assaying ProQ for RNA chaperone activity 185 Assaying NMB1681 for RNA chaperone activity 188 Materials and Methods 189 References 197 Appendix B: Biochemical characterization of the envelope stress accessory protein, CpxP Overview 199 Introduction 200 Results 202 Discussion 207 Materials and Methods 209 References 220 List of Tables Page Chapter 2 Table 2-1 - Effect of FinO mutations on rates of strand exchange, duplexing, and conjugative inhibition 76 Table 2-2 - FinO double alanine mutant primer oligonucleotides 77 Table 2-3 - FinO single alanine mutant primer oligonucleotides 78 Table 2-4 - DNA templates for in vitro transcription of RNAs for Chapter 2 79 Chapter 3 Table 3-1 - Data collection statistics for FinO 26-186 W36A/SLII guga-5 crystal collected at ALS 8.3.1 117 Table 3-2 - FinO truncation mutants Gateway attB PCR oligonucleotides 118 Table 3-3 - DNA templates for in vitro transcription of RNAs in Chapter 3 119 List of Figures Page Chapter 1 Figure 1-1 - The bacterial conjugation cycle 29 Figure 1-2 - Organization of the genes of the F-plasmid transfer operon and their functions 30 Figure 1 -3 - Summary of early fertility inhibition experiments 31 Figure 1-4 - Summary of the components of fertility inhibition 32 Figure 1-5 - Model of FinOP repression of bacterial conjugation 33 Figure 1-6 - Loop-loop interactions in antisense RNA regulatory systems 34 Figure 1-7 - Regulation of ColE1 DNA replication by RNA I and Rom 35 Figure 1-8 - The action of RNA chaperones 36 Figure 1 -9 - RNA chaperone activity of the HIV-1 nucleocapsid protein 37 Figure 1-10 - RNA chaperone activities of Hfq 38 Chapter 2 Figure 2-1 - RNA and protein constructs used in this study 68 Figure 2-2 - FinO can perform strand exchange on
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