Copyright by Meng Zhao 2006 The Dissertation Committee for Meng Zhao Certifies that this is the approved version of the following dissertation: Genetic and Biochemical Studies on the Differential Modulation of RNA Decay and Processing by Inhibitory Proteins in Escherichia coli Committee: George Georgiou, Supervisor Ian J. Molineux Charles F. Earhart, Jr. Vishwanath Iyer Richard J. Meyer Genetic and Biochemical Studies on the Differential Modulation of RNA Decay and Processing by Inhibitory Proteins in Escherichia coli by Meng Zhao, B.M., M.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin December, 2006 Dedication To my family, to whom I owe everything Acknowledgements First, I want to thank my advisor, George Georgiou, for being such a great mentor the past five (or so) years. Your trust in my ability, encouragement, support and guidance will never be forgotten. I am grateful to my committee members, Ian Molineux, Charles Earhart, Vishwanath Iyer, and Richard Meyer, for their interest in this work and helpful suggestions. I would like to thank Yasuaki Kawarasaki and Li Zhou for their assistance on the RraA and RraB regulation work; Junjun Gao, Kangseok Lee and Stanley Cohen, for their collaborations on the characterization of RraB function; Chainaya Jain for his suggestion on the design of genetic screen. I would also like to thank Aiko Umeda, Ashwini Devkota, and Jeff Chan, for their help on making the degradosome component expression constructs. I am grateful to Alan Lambowitz and Andy Ellington, who allowed me to use their labs for the RNA related experiments. I acknowledge Robert Simons, Charles Yanofsky, Andrew Emily, Sidney Kushner and Lin-Chao Sue for sharing strains and antibodies. I thank Danielle Tullman-Ercek and Clinton Leysath for proofreading this dissertation. Then there are so many other lab members –Karl Griswold, Andrew Hayhurst, Xiaoming Zhan, Lluis Masip, Laura Segatori, and Navin Varadarajan who all helped me so much with both research and keeping my sanity. And thanks also to everyone else who has made the lab such a fun place to work. Beyond the lab, special praise goes to Jo Frederick and her family. Thank you for introducing the love of God to me, which has changed my life. I would like to express my deepest appreciation to my parents and my sister for their unconditional love and support. And finally, my husband, Yibin, gets the most thanks for his love, understanding and support, over the last four and a half years. v Genetic and Biochemical Studies on the Differential Modulation of RNA Decay and Processing by Inhibitory Proteins in Escherichia coli Publication No._____________ Meng Zhao, Ph.D. The University of Texas at Austin, 2006 Supervisor: George Georgiou The regulation of mRNA decay is a critical post-transcriptional step in the control of gene expression. In E. coli, RNase E carries out the first and rate-limiting step in the decay of most mRNAs, as well as in the processing of ribosomal, transfer RNAs and small regulatory RNAs. The RNase E protein has two domains: the catalytic N-terminal half and the C-terminal half containing the scaffold region for the assembly of an RNA degrading machine termed the degradosome. Earlier studies in our lab identified the trans-acting proteins, RraA and RraB, which inhibit RNase E activity through direct- interaction with the enzyme. The present work explores several mechanistic, physiological and biotechnology- related aspects of the modulation of E. coli RNA decay and processing by inhibitory proteins. We found that, in contrast to RraA, RraB interacts with a different site on RNase E, results in distinct changes in degradosome composition, and interferes with cleavage of a different set of transcripts. Therefore, our results revealed a novel vi mechanism whereby a selective remodeling of the degradosome by endonuclease binding proteins serves to differentially regulate RNA decay and processing in E. coli. We developed protocols to express and purify the C-terminal truncated forms of RNase E. A system to reconstitute part of the degradosome and determine the consequences of RraA and RraB binding to the kinetics of RNA degradation in vitro was established. We showed that rraA is transcribed predominantly from its own promoter (PrraA) located in the menA - rraA intergenic region. Transcription from PrraA is σs-dependent and is induced upon entry into stationary phase. Moreover, the synthesis of RraA is regulated at the post-transcriptional level by RNase E, suggesting the existence of a feedback regulatory circuit between RraA and RNase E. RraB is transcribed from a promoter (PrraB) which is divergent from the argI promoter. Contrary to the previous data, we could not find any evidence that rraB promoter activity, mRNA abundance and protein level are affected by changes in tryptophan or arginine metabolism. In contrast to RraA, the expression of RraB does not change during different growth phases. vii Table of Contents Chapter 1 Introduction and Background ............................................................ 1 Post-transcriptional control of gene expression: mRNA decay in E. coli ..... 1 Ribonucleases control RNA decay and processing ............................ 2 5'-end events in mRNA decay .............................................................. 5 3'-end events in mRNA decay .............................................................. 6 Small regulatory RNAs......................................................................... 8 RNase E and the degradosome ...................................................................... 9 RNase E .............................................................................................. 9 Function of the degradosome ............................................................ 15 RNase E Action............................................................................................ 21 RNase E cleavage specificity.............................................................. 21 RNase E autoregulation ...................................................................... 26 RraA, the trans-acting factor modulating RNase E activity ........................ 26 Research outline........................................................................................... 30 Chapter 2 RraB: A second RNase E-inhibitory protein that differentially modulates the mRNA abundance ......................................................................................... 32 Introduction.................................................................................................. 32 Materials and methods ................................................................................. 34 Strains and plasmids ........................................................................... 34 β-galactosidase assays ........................................................................ 34 RNA work........................................................................................... 35 Microarray procedures........................................................................ 37 Surface plasmon resonance analysis................................................... 37 Pull-down assay .................................................................................. 37 SPA purification of degradosomes ..................................................... 38 Mass spectrometry .............................................................................. 39 viii Results.......................................................................................................... 40 Transcript-specific inhibition of the endonuclease activity of RNase E by RraB............................................................................ 40 RraA and RraB interact differently with the CTH domain of RNase E .......................................................................................... 45 Binding of RraA and RraB affects the degradosome composition..... 49 Discussion.................................................................................................... 53 Chapter 3 Reconstitution of Degradosomes and Kinetic Analysis of RNA Degradation in vitro .............................................................................................. 57 Introduction.................................................................................................. 57 Materials and methods ................................................................................. 59 Strains and plasmids ........................................................................... 59 Expression and purification of truncated versions of RNase E .......... 61 Production and purification of RraA, RraB, PNPase, RhlB and Enolase ......................................................................................... 63 RNA cleavage assay ........................................................................... 63 Results.......................................................................................................... 64 Purification of C-terminal truncated RNase E.................................... 64 Determination of the kinetic parameters of RNase E ......................... 67 Consequences of RraA and RraB binding to the kinetics of RNA degradation in vitro.................................................................... 71 Production and purification