TOWARDS UNDERSTANDING HELICASE AND CHAPERONE ACTIVITIES IN THE RNA DEGRADOSOME Zi Ran Shen Wolfson College Department of Biochemistry University of Cambridge This dissertation is submitted for the degree of Doctor of Philosophy January 2020 DECLARATION This thesis is the result of my own work and includes nothing which is the outcome of work done in collaboration except as declared in the Preface and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. This thesis is does not exceed 60,000 words excluding tables, footnotes, bibliography, and appendices. i ABSTRACT The E. coli RNA degradosome is a complex multi-enzyme machine that is central to the post- transcriptional regulation of the cell. Some of its functions include maturing and processing sRNA, rRNA, and tRNA, as well as degrading mRNA. The key components of the RNA degradosome include the endoribonuclease RNase E, the DEAD-box RNA helicase RhlB, the glycolytic enzyme enolase, and the phosphorolytic exoribonuclease PNPase. The degradosome has also been previously shown to associate with the RNA chaperone Hfq to form a small RNA guided machinery that targets defined transcripts. This thesis attempts to investigate several characteristics of the degradosome, including the importance of RhlB, the structure of a portion of the RNase E C-terminal domain that recruits enolase and helicase, and the association between Hfq, ChiX and the C-terminus of RNase E. The thesis also explores structural details regarding the small domain of RNase E involved in both RNA binding and oligomerisation and its relationship to the RNA-binding KH domains. Utilizing a point mutation in the DEAD box of RhlB, I have found increased RNA affinity to mutant RhlB, the potential structure of Hfq — an RNA chaperone — bound to the sRNA ChiX, and a structural correlation between the RNase E small domain and other KH domains. Preliminary models of Hfq and ChiX structure show a novel binding mode for class II sRNAs as the majority of ChiX associates with the distal face of Hfq. Bioinformatic studies reveal evolutionary roots between the KH domains and the RNase E small domain, supporting the hypothesis that the RNase E small domain may be involved in a novel mode of RNA binding and recognition. ii Word Template by Friedman & Morgan 2014 Morgan & Friedman by Word Template ACKNOWLEDGEMENTS I would like to thank many people who have helped me in my PhD — if I were to list them all this PhD will surely exceed 60,000 words in names alone. In the interest of brevity, I will list only those integral to my life in Cambridge but rest assured I am deeply grateful for everyone who has helped me along the way, however briefly. I’d like to first and foremost thank Professor Ben Luisi, one of the kindest and most supportive people I’ve ever met in my life. Without him I would’ve never set foot in Cambridge, let alone finish this thesis. I’d also like to thank my lab mate Tom Dendooven for his endless advice on cryo-EM for which I am forever grateful and perpetually in awe. I’d also like to thank my lab mates (special shoutout to Miao, Kotryna, and Heather, and Tai) for all of your friendship and support. Thank you kindly to my friends at Wolfson College who brought so much joy to my live over the last four years. A big thank you to my parents, who have selflessly provided me with love and education throughout my life. Last but certainly not least, I’d like to thank my partner Bas Monsewije for all the highlights of my Cambridge experience — he continues to make me happier even though I am the happiest I’ve ever been. Thank you to everyone who’s been a part of this amazing journey with me. iii Towards Understanding Helicase and Chaperone Activities in the RNA Degradosome CONTENTS 1 INTRODUCTION ...................................................................................................... 12 1.1 OVERVIEW ............................................................................................................. 12 1.2 RNA DEGRADOSOME EVOLUTION AND ORGANIZATION ........................................ 19 1.2.1 RNase E 22 1.2.2 RNA helicase B 26 1.2.3 Enolase 29 1.2.4 Polynucleotide Phosphorylase 30 1.3 HFQ STRUCTURE AND FUNCTION ........................................................................... 33 1.4 SMALL REGULATORY RNAS: RYHB, SGRS, AND CHIX ......................................... 41 2 RHLB ACTIVITY IMPACTS E. COLI GROWTH ............................................... 52 2.1 INTRODUCTION ....................................................................................................... 52 2.2 RESULTS ................................................................................................................. 56 2.3 DISCUSSION ............................................................................................................ 67 2.4 MATERIALS AND METHODS.................................................................................... 70 2.4.1 Lambda Red recombination 70 2.4.2 Cell growth 71 3 BIOPHYSICAL AND PHYSIOLOGICAL ANALYSES OF RHLB E166Q MUTANT 72 3.1 INTRODUCTION ....................................................................................................... 72 3.2 RESULTS ................................................................................................................. 74 4 Zi Ran Shen - January 2020 Chapter 1: Introduction 3.2.1 Expression and purification of the ternary complex of RhlB, enolase and the recognition segment of RNase E 74 3.2.2 RNA binding 76 3.2.3 UV crosslinking and pulldown 78 3.3 DISCUSSION ........................................................................................................... 81 3.4 MATERIALS AND METHODS .................................................................................... 83 3.4.1 Cloning 83 3.4.2 Expression and Purification of ternary complex (RhlB + RNase E 603-850 + enolase) 86 3.4.3 MalEF in vitro transcription 88 3.4.4 Titration of MalEF by ternary complex (RhlB + RNase E 603-850 + enolase) 90 3.4.5 UV irradiation and pulldown 90 4 STRUCTURAL STUDIES OF HFQ BOUND TO CHIX ...................................... 92 4.1 INTRODUCTION ...................................................................................................... 92 4.2 RESULTS .............................................................................................................. 102 4.3 DISCUSSION ......................................................................................................... 111 4.4 METHODS ............................................................................................................ 115 4.4.1 Hfq purification 115 4.4.2 ChiX IVT 117 4.4.3 Supercomplex formation and crosslinking 117 4.4.4 Grid preparation 118 4.4.5 Data collection 118 4.4.6 Data processing 119 Zi Ran Shen - January 2020 5 Towards Understanding Helicase and Chaperone Activities in the RNA Degradosome 5 TOWARD UNDERSTANDING HOW SMALL RNAS ARE RECOGNISED BY RNASE E IN THE DEGRADOSOME ..................................................................... 121 5.1 INTRODUCTION ..................................................................................................... 121 5.2 RESULTS ............................................................................................................... 126 5.3 DISCUSSION .......................................................................................................... 131 5.4 MATERIALS AND METHODS.................................................................................. 134 6 SUMMARY AND PERSPECTIVES ..................................................................... 135 7 REFERENCES ......................................................................................................... 146 6 Zi Ran Shen - January 2020 Chapter 1: Introduction LIST OF TABLES TABLE 1: LIST OF STRAINS USED IN THIS STUDY 57 TABLE 2: LAG TIME, MAXIMUM DOUBLING TIME, ENTRY INTO STATIONARY PHASE/DEATH PHASE OF EACH STRAIN IN ALL CONDITIONS (MIN) 65 TABLE 3: LIST OF PRIMERS USED IN STRAIN CONSTRUCTION 70 TABLE 6: UV CROSSLINKING AN PULLDOWN OF RHLB 79 TABLE 7: AN OVERVIEW OF THE CLONING PROCESS AND ITS ASSOCIATED SEQUENCES 84 TABLE 8: LIST OF BUFFERS USED FOR TERNARY COMPLEX PURIFICATION 87 TABLE 9: LIST OF PRIMERS USED IN MALEF IVT 89 TABLE 10: LIST OF BUFFERS USED IN UV IRRADIATION AND PULLDOWN 91 Zi Ran Shen - January 2020 7 Towards Understanding Helicase and Chaperone Activities in the RNA Degradosome LIST OF FIGURES FIGURE 1: MECHANISM OF OMPF REPRESSION BY MICF 13 FIGURE 2: SCHEMATIC OF THE THREE MOST COMMON SRNA FUNCTIONS 14 FIGURE 3: HFQ AND ITS RNA BINDING MODES 16 FIGURE 4: THE ORGANIZATION OF VARIOUS KINDS OF DEGRADOSOMES 19 FIGURE 5: STRUCTURE OF THE N-TERMINAL CATALYTIC DOMAIN OF RNASE E 22 FIGURE 6: RHLB AND DEAD BOX HELICASE STRUCTURAL ORGANIZATION 26 FIGURE 7: ENOLASE AND ITS ASSOCIATIONS WITH THE E. COLI RNA DEGRADOSOME 29 FIGURE 8: PNPASE ORGANIZATION, RNASE E AND RNA BINDING 30 FIGURE 9: