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Defining the Constellation of RNA Elements That Associate with Bacillus Subtilis

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Defining the Constellation of RNA Elements That Associate with Bacillus Subtilis DEFINING THE CONSTELLATION OF RNA ELEMENTS THAT INTERACT WITH BACILLUS SUBTILIS HFQ APPROVED BY SUPERVISORY COMMITTEE Wade C. Winkler, Ph.D. Russell Debose-Boyd, Ph.D. Kevin H. Gardner, Ph.D. Benjamin Tu, Ph.D. DEFINING THE CONSTELLATION OF RNA ELEMENTS THAT ASSOCIATE WITH BACILLUS SUBTILIS HFQ By MICHAEL DAVID DAMBACH DISSERTATION /THESIS Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas August, 2012 ACKNOWLEDGEMENTS First and foremost, I would like to thank my mother Karen and my father David, who pushed me to chase my dreams where ever they may lead. Their love and support was a guiding light through many dark times. For without them none of this would have been possible. I would also like to bestow my gratitude upon my sister Megan and my Brother Mark, their encouragement and advice was critical to my completion of this thesis. I would like to extend my sincere thanks to past and present members of the Winkler lab; in particular Dr. Arati Ramesh, Dr. Irnov, and Dr. Catherine Wakeman. I have never worked in a more fun, stimulating, and enriching environment than the one I shared when we were all together. I wish you all the very best in your future endeavors. In addition, I would like to thank my thesis committee- Dr. Russell Debose-Boyd, Dr. Kevin Gardner, and Dr. Benjamin Tu for taking the time to participate in my studies as well as offering critical advice and guidance through this process. I must extend my sincerest thanks and gratitude to Drs. Melanie Cobb and Steve McKnight. I cannot put into words the lessons that you have bestowed on me. I would not be the scientist or the person I am today without your guidance and leadership and for that I am eternally grateful. And finally I must give the ut most thanks and gratitude to my mentor Dr. Wade Winkler. Wade took me in and nurtured me when I was at my lowest point during this process. His unconditional support and encouragement fostered and stoked my scientific passion and reminded me why we love this endeavor we call science. I can honestly say I’m a better person for knowing him and his family. His teachings transcend science and I will carry them with me on the journey through life. Copyright By MICHAEL DAVID DAMBACH, 2012 All Rights Reserved DEFINING THE CONSTELLATION OF RNA ELEMENTS THAT ASSOCIATE WITH BACILLUS SUBTILIS HFQ Michael David Dambach, Ph.D The University of Texas Southwestern Medical Center at Dallas, 2012 Wade C. Winkler, Ph.D Bacteria utilize a wide variety of genetic regulatory strategies in order to sense and respond to various environmental fluctuations in nutrient availability, temperature, salinity, and oxygen among others. As such, bacterial species have evolved highly coordinated and tightly regulated systems as a means of vi efficiently responding to potentially deleterious changes in environmental conditions. Traditionally DNA binding transcriptions factors were thought to be the primary means by which the cell executes a selective genetic response. However, the advent of microarray and next generation sequencing platforms, coupled with the wealth of sequenced genomes and powerful bioinformatics have revealed that RNA mediated post transcriptional gene regulation is wide spread in bacterial species and may in fact rival protein based regulatory systems in scope and breadth. RNA mediated post transcriptional gene regulation is broadly divided into two categories-those in which the RNA element is transcribed with the mRNA it regulates (cis-acting regulatory RNAs) or those which are transcribed independently from the gene that they regulate (trans-acting regulatory RNAs). In general cis-acting RNA elements are embedded within a 5’ UTR of a gene that they regulate and may or may not require a protein cofactor to execute genetic regulation. Whereas, trans-acting regulatory RNAs, also known as sRNAs, function via base pairing with their target mRNA and this usually requires the protein chaperone Hfq. Hfq mediated gene regulation is poorly understood in Gram-positive organism, thus I undertook studies of this protein in the model Gram-positive organism Bacillus subtilis. I used co-immunoprecipitation and vii deep-sequencing to define the suite of RNA elements that associate with this regulatory protein. In addition I performed global transcriptomic studies on an Hfq deletion mutant in order to identify genes that are regulated via Hfq. These studies identified sRNAs that may be involved with sporulation. This led me to analyze the transcriptomic profile of Bacillus subtilis spores in an attempt to identify new sRNA regulators. viii TABLE OF CONTENTS ACKNOWLEDGEMENTS..........................................................................................iii ABSTRACT...............................................................................................................vi TABLE OF CONTENTS..............................................................................................ix LIST OF FIGURES....................................................................................................xv LIST OF TABLES.....................................................................................................xxi LIST OF ABBREVIATIONS.....................................................................................xxii CHAPTER ONE Introduction and literature Review…………………............................1 Cis-encoded regulatory RNAs......................................................................1 Control of transcription termination...............................................3 Control of translation initiation.......................................................4 Control of mRNA stability................................................................5 Control of eukaryotic splicing..........................................................6 ix Trans-encoded regulatory RNAs.................................................................8 sRNA mediated translational repression.......................................11 The role of Hfq and sRNA binding.................................................12 Hfq binding and RNA stability.......................................................15 Antisense small RNA regulators....................................................18 Small RNA mediated protein regulation.......................................19 Regulation of RNA binding proteins..............................................19 Regulation of enzyme activity.......................................................21 CHAPTER TWO Identification of RNA elements that associate with B. subtilis Hfq in vivo....................................................................................................................28 Introduction..............................................................................................28 Results.......................................................................................................34 Expression of Hfq in B. subtilis......................................................34 General approach for identifying B. subtilis regulatory RNAs.......37 x mRNAs and mRNA coding region fragments that associate ........41 Long mRNA leader regions associate with Hfq.............................43 sRNA regulators that associate with Hfq.......................................48 Concluding remarks and discussion..........................................................54 CHAPTER THREE Analysis of the consequence of loss of Hfq on the global transcriptional landscape of Bacillus subtilis Hfq ...............................................102 Introduction............................................................................................102 Results and discussion............................................................................103 Phenotype of Hfq deletion mutant.............................................103 Global transcriptomic profile of an Hfq deletion mutant............114 sRNA abundance and deletion of Hfq.........................................116 Hfq and ORF’s..............................................................................120 Hfq and start codons...................................................................122 Internal Peaks..............................................................................123 xi Hfq and 5’ leader sequences.......................................................125 Discussion and future directions.............................................................127 CHAPTER FOUR Identification of sporulation specific small RNAs and the Examination of the Global Transcriptome of Bacillus Subtilis spores.................152 Results.....................................................................................................152 CsfG is a fore spore specific sRNA...............................................152 mRNA expression in spores.........................................................157 sRNA expression in spores..........................................................161 Discussion and future directions.............................................................168 CHAPTER FIVE Materials and Methods..............................................................201 Co-Immunoprecipitation of Hfq..............................................................201 Preparation of samples
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