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Role of Curved DNA in Promoter Recognition with Bacillus Subtilis and Escherichia Coli RNA Polymerase

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Role of Curved DNA in Promoter Recognition with Bacillus Subtilis and Escherichia Coli RNA Polymerase Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1994 Role of Curved DNA in Promoter Recognition With Bacillus Subtilis and Escherichia Coli RNA Polymerase. Cheryl Anne Nickerson Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Nickerson, Cheryl Anne, "Role of Curved DNA in Promoter Recognition With Bacillus Subtilis and Escherichia Coli RNA Polymerase." (1994). LSU Historical Dissertations and Theses. 5747. https://digitalcommons.lsu.edu/gradschool_disstheses/5747 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. 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Ann Arbor, MI 48106 ROLE OF CURVED DNA IN PROMOTER RECOGNITION WITH Bacillus subtilis AND Escherichia coli RNA POLYMERASE A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirement for the degree of Doctor of Philosophy in The Department of Microbiology by Cheryl Anne Nickerson B.S., Tulane University, 1983 M.S., University of Missouri, Columbia, 1988 May, 1994 DEDICATION This work is dedicated to my parents, Dr. and Mrs. Max A. Nickerson. It has only been through their constant love, support, encouragement, and unwaivering belief in my abilities, that I have found the strength necessary to meet any challenges that have faced me. I can state without hesitation that had it not been for their patience and understanding, this degree could never have been realized. ACKNOWLEDGEMENTS I would like to express my sincere appreciation to my major professor, Dr. Eric C. Achberger, for providing me not only with the laboratory facilities necessary to conduct my research but also the opportunity to intellectually challenge myself as a scientist. His advice as a mentor and colleague is highly valued and will serve me well in the future. Sincere gratitude is also extended to Dr. John Battista for his encouragement and invaluable advice and insight into my scientific pursuits both here at L.S.U. and in future endeavors. He was always very supportive and generous with his time. Special thanks to Dr. H.D. Braymer, who was always very helpful and encouraging, and genuinely interested in my future as a scientist. My thanks also to Dr. Randall Gayda and Dr. Ding Shih for their advice, patience, and constructive criticism of this dissertation. Their generous donation of time in this effort was greatly appreciated. I would also like to extend my deepest appreciation and gratitude to my fellow graduate students in the microbiology department for their friendship and encouragement. They helped make my stay at L.S.U. an enjoyable one. Special thanks to my dear friend, Anand Immaneni, whose companionship, generosity, and loyalty will never be forgotten. My fellow lab colleague, Doug Stemke, was always very patient and helpful, his kindness was most appreciated. My thanks also to Evie Vandergrifft, for her valued friendship and unique outlook on the "graduate experience", to Tim Beary, for his helpful advice and encouragement, and Mark Ott, for his patience, support, and friendship. My heartfelt thanks are also extended to Saurabh Singh, Karuna Murray, Marilyn Morse, Mena Cho, Kevin Riley, Michael Raggio, Dr. C.N. Riley, and Dr. C.A. Buddy, for their assistance, patience and support. Their friendship and unselfish donation of time made my job much easier. I would also like to extend my deepest appreciation to my wonderful friend, Gopal Hara. His support and encouragement were most appreciated, and his courage admired. A special acknowledgement to my grandparents, Mr. and Mrs. Raymond Bartee, Mr. and Mrs. I.J. Nickerson and to my uncle Mr. Herald Bartee. As with all endeavors in my life, they have always supported, encouraged and inspired me to do my best. Finally, and most important of all, I wish to thank my parents for their constant love, support and encouragement throughout my life. Had it not been for them, I could never have found the strength, determination, and fortitude necessary to pursue this degree. TABLE OF CONTENTS Page Dedication .............................................................................................................. ii Acknowledgements .................................................................................................. iii List of Tables ........................................................................................................ vii List of Figures ....................................................................................................... viii Abstract ................................................................................................................... x Introduction .............................................................................................................. 1 RNA polymerase ......................................................................................... 1 Promoters ..................................................................................................... 6 Regulatory molecules ...................................................................................9 DNA supercoiling .........................................................................................13 Transcription initiation kinetics .................................................................. 14 Sequence-dependent DNA curvature ....................................................... 16 Experimental Procedures .................................................................................... 26 Bacterial strains and plasmids .................................................................. 26 Media, growth conditions and DNA transformations ............................ 27 Construction of \p K and XpL-derived promoters ..................................... 28 Construction of transcriptional fusions between lambda-derived promoters and the cat gene .............................. 31 Plasmid isolation ..........................................................................................32 Expression and detection of chloramphenicol acetyltransferase ............................................................................... 32 RNA polymerase isolation ...........................................................................33 Nitrocellulose filter binding assay ........................................................... 34 Gel retardation analysis ................................................................................ 36 DNasel footprint analysis ........................................................................... 37 DNA fragment electrophoretic mobility determinations ...........................40 Materials ..................................................................................................... 40 v Results 42 Electrophoretic mobilities of promoter-containing DNA fragments ............................................................................... 44 CAT expression of the X-derived promoter constructs ......................... 46 Relative affinity of RNA polymerase for promoters using the filter binding assay ....................................................... 50 Gel retardation analysis of the relative affinity of RNA polymerase for linear and supercoiled molecules ....................... 56 DNasel footprinting analysis of the interactions of E. coli RNA polymerase with the XpR-derived promoters ............................... 65 Discussion .......................................................
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