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CHARACTERIZATION of the Metk and Yitj LEADER Rnas from THE CHARACTERIZATION OF THE metK AND yitJ LEADER RNAs FROM THE Bacillus subtilis S BOX REGULON DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Vineeta A. Pradhan, B.S. Graduate Program in Microbiology The Ohio State University 2012 Dissertation Committee: Professor Tina M. Henkin, Advisor Professor Charles J. Daniels Professor Kurt L. Fredrick Professor Joseph A. Krzycki Copyright by Vineeta A. Pradhan 2012 ABSTRACT A variety of mechanisms that regulate gene expression have been uncovered in bacteria. Riboswitches are cis-acting regulatory sequences that reside typically in the untranslated regions of bacterial mRNAs. Riboswitches serve as genetic regulatory switches that sense and respond specifically to environmental signals to regulate expression of the downstream gene, typically in the absence of any protein factor. The S box riboswitch is a transcription termination control system found mostly in Gram- positive bacteria that regulates the expression of many genes involved in sulfur metabolism. The S box genes are characterized by the presence of a set of highly conserved primary sequence and secondary structural elements in the untranslated leader region upstream of the regulated coding sequence. SAM, the molecular effector of the S box riboswitch, is synthesized from methionine and ATP. Expression of the majority of the S box genes is induced during methionine starvation (when SAM pools are low) and is repressed in the presence of methionine (when SAM pools are high). In spite of high sequence and structural conservation, a few S box leader RNAs from Bacillus subtilis fail to exhibit typical S box gene regulation and variation is seen in response to SAM both in vivo and in vitro. This work examines the leader RNA elements that contribute to the observed S box variability, with a special focus on the metK leader ii RNA. Investigation of the metK leader RNA was performed using biochemical and genetic techniques. We modulated in vivo SAM pools without removing methionine from the growth medium and provided evidence for a SAM-dependent change in metK gene expression in vivo. Phylogenetic analyses revealed the presence of unique sequence elements, the Upstream (US) and Downstream (DS) boxes, that are highly conserved in the metK leader RNAs in several Firmicutes. Using RNase H assays, we showed that these regions are involved in a base-pairing interaction that is stabilized in the absence of SAM. Extensive mutagenic analysis of the US and DS box sequences confirmed the need for an intact US-DS base-pairing interaction for response to SAM in vivo. Transcript stability and abundance studies showed that the US-DS pairing is disrupted in the presence of SAM and that any alteration in the US box sequence reduces transcript stability significantly. A model for metK regulation was proposed in which the metK gene is regulated at the level of mRNA stability, in addition to being under the control of the S box regulon. In vitro investigation of the B. subtilis yitJ SAM binding pocket was conducted to identify RNA determinants for ligand affinity and specificity. We attempted to generate yitJ variants that exhibit higher SAM affinity compared to wild-type yitJ or a change in ligand specificity. Extensive mutational analysis was conducted as part of the crystal structure study of the B. subtilis yitJ RNA in complex with SAM and mutants were tested for effects on in vitro transcription and SAM binding. As expected, most of the mutants exhibited loss of SAM binding. However, some mutants resulted in constitutive high iii termination, indicating that the RNA was locked in a SAM-bound-like conformation in the absence of SAM. Selected mutants were also tested in response to a series of SAM analogs and compared to the response of the wild-type yitJ RNA. Individual RNA elements critical for S box riboswitch function were examined using the metE and yusC leader RNAs. Our data suggest that both the SAM-binding domain and the terminator/antiterminator structures play a crucial role in the calibration of the S box regulatory system. The effect of the metK promoter and US box sequence on expression of yusC was also examined. We predict that the metK promoter, along with the US box sequence, are responsible for reduced transcription initiation or reduced RNA polymerase processivity in vitro. These studies provide possible implications of the metK promoter and US box sequence on transcription and therefore metK regulation. iv This work is dedicated to my parents Neela and Prakash Kurlekar. v ACKNOWLEDGEMENTS I wish to express my sincere gratitude to my advisor, Dr. Tina Henkin, for her guidance, patience, and support throughout the years. It has been a privilege to work with such a gifted scientist and excellent mentor. I am grateful to Dr. Frank Grundy for his guidance and support throughout the years. Dr. Grundy played an instrumental role in this project, particularly in the work of the metK project. I would like to extend a special thanks to my committee members, Dr. Charles Daniels, Dr. Kurt Fredrick and Dr. Joseph Krzycki, for their guidance, support and time over the years. I also wish to thank my colleague and dear friend, Dr. Sharnise N. Mitchell, for being a great support both on a scientific and personal level, throughout the years. I really appreciate and value her friendship, guidance, and constant encouragement. I would like to thank the present and past members of the lab, particularly, Dr. Brooke A. McDaniel, Dr. Jerneja Tomšič and Dr. Enrico Caserta for their helpful discussions and encouragement during our time together. I would also like to thank Susan Tigert and Chris Woltjen for their technical assistance. I would like to thank Mike Zianni from the Plant Microbe and Genome Facility vi for his help with the qRT-PCR assays. I wish to express my special thanks to Dr. Madhura Pradhan for her constant encouragement and support throughout the years. Most of all, I am truly grateful to my family, especially my husband Ashish and my parents Neela and Prakash Kurlekar. Without their encouragement, patience and support this would not have been possible. vii VITA January 19, 1982 ............................................Born – New Brunswick, New Jersey 2003................................................................B.S., Microbiology, University of Pune 2004-present ...................................................Graduate Teaching and Research Associate, Department of Microbiology, The Ohio State University PUBLICATIONS 1. McDaniel BA, Grundy FJ, Kurlekar VP, Tomsic J, Henkin TM. 2006. Identification of a mutation in the Bacillus subtilis S-adenosylmethionine synthetase gene that results in derepression of S box gene expression. J Bacteriol 188: 3674-3681. 2. Lu C, Ding F, Chowdhury A, Pradhan V, Tomsic J, Holmes WM, Henkin TM, Ke A. 2010. SAM recognition and conformational switching mechanism in the Bacillus subtilis yitJ S box/SAM-I riboswitch. J Mol Biol 404: 803-818. FIELDS OF STUDY Major Field: Microbiology viii TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii DEDICATION .................................................................................................................... v ACKNOWLEDGEMENTS ............................................................................................... vi VITA ................................................................................................................................ viii LIST OF TABLES ............................................................................................................ xv LIST OF FIGURES ......................................................................................................... xvi LIST OF ABBREVIATIONS ........................................................................................... xx CHAPTER 1 ....................................................................................................................... 1 REGULATION OF GENE EXPRESSION BY RIBOSWITCHES ........................... 1 1.1 Types of riboswitch classes .............................................................................. 7 1.2 Riboswitch classes ......................................................................................... 10 1.2.1 RNA Thermosensors ................................................................................. 10 1.2.2 T box riboswitch ....................................................................................... 15 1.2.3 Amino acid binding riboswitches ............................................................. 19 1.2.3.1 L box riboswitch ................................................................................ 19 1.2.3.2 Glycine riboswitch ............................................................................. 21 1.2.3.3 Glutamine riboswitch ......................................................................... 24 1.2.4 Purine-sensing riboswitches..................................................................... 26 ix 1.2.4.1 Guanine riboswitch ........................................................................... 27 1.2.4.2 Adenine riboswitch ........................................................................... 28 1.2.4.3 2’-Deoxyguanosine riboswitch ........................................................ 30 1.2.4.4 PreQ1 riboswitch ..............................................................................
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