ii Dedication This dissertation is the culmination of all the sacrifices done by my loving parents Vasuki Shanmuganatham and Shanmuganatham and for that I dedicate this dissertation to them. iii Acknowledgements My heartfelt thanks goes to my mentor Dr. Martha M. Howe for providing me with an opportunity to work in her lab and giving her guidance and input in all my scientific endeavors. I am especially grateful to her for her unwavering support in my crystallography project and letting me

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... from my own mistakes. I am grateful to my committee member Dr. Hee Won Park who has been a co-mentor, friend and continuous source of encouragement for me throughout my crystallography project. I would also like to thank my other committee members, Dr. Marko Radic, Dr. Rajendra Raghow, and Dr. James Patrick Ryan for their suggestions, help and advice during the course of my graduate study. I also wish to thank Ms. Manimekalai Ravichandran for being very tenacious and helping me crystallize the complex used in this dissertation. I personally thank my best friends Jeetendra and Muthiah for guiding me through my tumultuous graduate study and to their invaluable friendship and support in my personal and public life. I would also like to thank my adoptive sister Ms. Himangi Jayakar for her continuous support and encouragement. I am very thankful to my girlfriend Ms. Angi Beau who has been at my side with words of encouragement and motivation whenever I needed her for the good latter part of my graduate study. Words cannot describe how to thank the two most influential people in my life, my parents Vasuki and Shanmuganatham. This dissertation would not be possible but for their love, and unwavering support in my abilities. In addition, I would like to give them my heartfelt thanks for all their sacrifices they have made for me. iv Abstract The lytic cycle of phage Mu is regulated by a transcriptional cascade consisting of early, middle and late transcription. The Mor protein is an activator of the middle promoter P m and is encoded by the last gene of the early transcript. The C protein is an activator of the four late promoters P lys , P I , P P , and P mom and is expressed from the middle transcript. Both Mor and C proteins bind an imperfect dyad-symmetry element just upstream and overlapping the -35 region of P m and P lys respectively. The main aims of this study was, (1) To understand the binding specificity of C and determine a possible consensus sequence for C binding, and (2) To crystallize the C : DNA complex as a first step towards structure determination. In previous work, single base substitution mutations in P lys identified bases and positions important for C binding and activation. To get a consensus sequence for C binding, we tested additional candidate mutations within and flanking the C binding sequence. Wild-type C protein was used in gel mobility shift assays with annealed oligonucleotides containing mutations, insertions and deletions. The assay showed that, (1) mutation in positions -53, -52 and -32 did not affect C binding, (2) mutations flanking the IR spacer (-40, -41, -46, -47) influence C binding, and (3) insertion or deletion of a single base pair in the IR spacer abolished C binding. Mor and C proteins are the founding members of a new class of transcription factors. The Mor structure revealed that it has a classical DNA-binding HTH motif and a dimerization domain. Based on the structure it has been proposed that Mor has to undergo conformational changes to bind DNA. Modelling of C based on the Mor v structure revealed that C might also have a dimerization domain and a HTH DNA binding motif. To see if any conformational changes occur in C when it binds DNA, cocrystallization of a C : DNA complex was undertaken. Preliminary structural analysis of the complex revealed that under the crystallization conditions used C protein is bound to its symmetrical binding site using two HTH motifs from two C dimers without inducing any conformational change in itself or the DNA.