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Functional Analysis of Ribonuclease Iii Regulation by A FUNCTIONAL ANALYSIS OF RIBONUCLEASE III REGULATION BY A VIRAL PROTEIN KINASE ------------------------------------------------------------------------------------------------------------ A Dissertation Submitted to the Temple University Graduate Board ------------------------------------------------------------------------------------------------------------ in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ------------------------------------------------------------------------------------------------------------ by Swapna Gone Jan, 2012 Examining Committee Members: Dr. Allen W. Nicholson, Advisory Chair, Department of Chemistry Dr. Rodrigo B. Andrade, Department of Chemistry Dr. Michael J. Zdilla, Department of Chemistry Dr. Frank Chang, External Member, Department of Biology i ABSTRACT FUNCTIONAL ANALYSIS OF REGULATION OF RIBONUCLEASE III BY A VIRAL PROTEIN KINASE Swapna Gone Doctor of Philosophy Temple University, 2011 Doctoral Advisory Committee Chair: Dr. Allen W. Nicholson The bacteriophage T7 protein kinase enhances T7 growth under suboptimal growth conditions, including elevated temperature or limiting carbon source. T7PK phosphorylates numerous E. coli proteins, and it has been proposed that phosphorylation of these proteins is responsible for supporting T7 replication under stressful growth conditions. How the phosphorylation of host proteins supports T7 growth is not understood. Escherichia coli (Ec) RNase III is phosphorylated on serine in bacteriophage T7-infected cells. Phosphorylation of Ec-RNase III induces a ~4-fold increase in catalytic activity in vitro. Ec-RNase III is involved in the maturation of several T7 mRNAs, and it has been shown that RNase III processing controls the translational activity and stability of the T7 mRNAs. Perhaps T7PK phosphorylation of Ec-RNase III ensures optimal processing of T7 mRNAs under suboptimal growth conditions. In this study a biochemical analysis was performed on the N-terminal portion of the 0.7 gene (T7PK), exhibiting only the protein kinase activity. In addition to phosphotransferase activity, T7PK also undergoes self-phosphorylation on serine, which down-regulates catalytic activity by an unknown mechanism. Mass spectral analysis revealed that Ser216 is the autophosphorylation site in T7PK. The serine residue is highly conserved, which in turn suggests that autophosphorylation is a conserved reaction with functional importance. Phosphorylated T7PK exhibits reduced phosphotransferase activity, compared to its dephosphorylated counterpart (dT7PK). The dT7PK exhibits ii enhanced ability to phosphorylate proteins, as well as undergo autophosphorylation. The mechanism by which autophosphorylation inhibits T7PK activity is unknown. An in vitro phosphorylation assay revealed that T7PK directly phosphorylates RNase III. Ec-RNase III processing activity is stimulated from two to ten-fold upon phosphorylation by the T7PK. The primary site of phosphorylation in RNase III is found to be Ser33, and Ser34 may act as the recognition determinant for T7PK. This was established by Ser →Ala mutations at the concerned site. The enhancement of catalytic activity is primarily due to a larger turnover number (kcat), with some additional contribution from a greater substrate binding affinity, as revealed by lower Km and K‟D values. Substrate cleavage assays under single turn over conditions established that the product release is the rate limiting step. Since there is no significant increase in the kcat as measured under single-turnover (enzyme excess) conditions, the increase in the kcat in the steady-state is due to enhancement of the product release step, and not due to an enhancement of the hydrolysis (chemical) step. iii To my dear parents, brother and husband for their love and support iv ACKNOWLEDGEMENTS It‟s been a long journey and most of it would not have been possible without the help and kindness of many people in my life. I hope I do not forget anyone but incase I do, I‟m sorry and thank you for all you have done for me so that I could come this far. May be the most important decision in graduate school was to choose my research advisor and I‟m glad I have chosen Dr. Allen W.Nicholson for his able guidance. He has been a constant source of inspiration and motivation, and also helped me inculcate the passion for science I have today. His doors were always open for us and he patiently answered all our questions. I was never afraid to show him negative results and he never failed to appreciate my success in research. He let me explore my own ideas and learn from my failures. Thank you Dr.Nicholson, for giving me the gift of knowledge and independent thinking. My sincere thanks to my committee members, Dr. Michael Zdilla, Dr.Rodrigo Andrade and Dr.Frank Chang for helping me out with my thesis. I would also like to thank Dr. William S. Brinigar who taught me the skills to work in the lab. I am thankful to Dr. Robert J. Stanley for his guidance through the initial years of my graduate school. I am very grateful to Dr. Rhonda Nicholson for her encouragement and advice. Many thanks to the lab members Dr. Lilian Nathania, Ritu Jaggi, Dr.Zhongjie Shi, Samridhi Paudhyal and Shiv Redhu for their help in the lab, acquaintance, support and valuable discussions. I would also like to thank Dr. Lee. Aggison and Dr. Prerna Sharma, whom I never met, but contributed to this project. I am thankful to the Department of Chemistry, Temple University and Dr. Nicholson for their financial support through teaching and research assistantships respectively. I would also like to thank Dr. Findeison for making my teaching experience at Temple University enjoyable. I also wish to express my appreciation to my friends, Dr.Sudha Chennasamudram, Dr.Deepa Rapolu, Dr. Venkat Velvadapu, Dr.Chandni Vyas, Swapnil, Dr.Gautam Kodali, Dr. Salim Siddiqui, Aditya, Shiva and Dr.Ananth Reddy for their encouragement, support v and patience. Sudha and Deepa deserve a special mention for their support and motivation. This PhD was made into a reality by my wonderful parents Satyanarayana Rao and Padmavathi Gone and my ever encouraging brother Dr. Santosh Gone. I cannot thank them enough for the sacrifices they have made so that I could see this day in my life. Thanks dad for having faith in me and thanks amma for always being so optimistic. This PhD would not have ever started without your guidance and motivation anna, and I thank you for that. This PhD was your dream too, hope I made you proud. My deepest thanks to my best friend and husband, Sharvan Daggula who has been very supportive and encouraging throughout the process. Thanks for understanding my stress levels and frustrations. Not enough words can express how much I appreciate the sacrifices and the choices you made for me and my PhD. To all of you, I warmly appreciate your generosity, understanding and encouragement. vi TABLE OF CONTENTS ABSTRACT ................................................................................................................... ii DEDICATION ............................................................................................................... iv ACKNOWLEDGEMENTS ............................................................................................. v LIST OF FIGURES ......................................................................................................... x LIST OF TABLES ......................................................................................................... xi LIST OF SCHEMES .....................................................................................................xii CHAPTER 1. INTRODUCTION ....................................................................................................... 1 1.1 Biological roles of RNA ................................................................................ 2 1.2 RNA processing and Ribonuclease III ........................................................... 4 1.2.1 Classification of RNase III family members .............................. 5 1.2.2 Eukaryotic RNase III structures and functions ........................... 6 1.3 Bacterial Ribonuclease III ........................................................................... 16 1.3.1 Ribonuclease III structure and mechanism............................... 22 1.3.2 Crystal structure of bacterial RNase III .................................... 27 1.3.3 Proposed reaction pathway for RNase III ................................ 29 1.4 Protein Phosphorylation .............................................................................. 32 1.4.1 Classification of protein kinases .............................................. 34 1.4.2 Role of protein phosphorylation .............................................. 35 1.4.3 Autophosphorylation of protein kinases................................... 37 1.5 Bacteriophage T7 structure and strategy of infection ................................... 40 1.5.1 T7 protein kinase..................................................................... 44 1.5.2 T7PK in T7 mRNA processing by RNase III ........................... 46 1.6 Specific aims ............................................................................................. 50 vii 2. MATERIALS AND METHODS ............................................................................... 51 2.1 Materials and Reagents .............................................................................. 52 2.1.1 Materials ...............................................................................
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