Faculty of Science and Technology A functional and structural study of three bacterial nucleic acid-interacting proteins The story of a Ferric Uptake Regulator, an Oligoribonuclease and an ATP-dependent DNA ligase Kristel Berg A dissertation for the degree of Philosophiae Doctor – November 2019 A functional and structural study of three bacterial nucleic acid-interacting proteins The story of a Ferric Uptake Regulator, an Oligoribonuclease and an ATP-dependent DNA ligase Kristel Berg A dissertation for the degree of Philosophiae Doctor Department of Chemistry Faculty of Science and Technology November 2019 To Terje, Malvin and Sigrid Acknowledgments This work was carried out at the Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, Faculty of Science and Technology at The University of Tromsø - The Arctic University of Norway. Financial support was granted by the UiT – The Arctic University of Norway. I would also like to acknowledge the financial support and training from BioStruct, the Norwegian Graduate School in Structural Biology and BioCat, the Norwegian Graduate School in Biocatalysis. My sincerest gratitude goes to my main supervisor Ingar Leiros for giving me the opportunity to work on this project. I highly appreciate the encouragement, humour, and great patience I received from you all these years and I feel grateful to have had such a supportive, understanding and positive supervisor throughout this journey’s ups and downs. Your impressive scientific knowledge has been valuable, and I have learnt a lot from you. Thank you also for the immense amount of time and effort you have brought into finalizing the papers and this thesis. It has been a pleasure to have Hege Lynum Pedersen as my co-supervisor from day one. You have provided me great support and encouragement, both work-related and in life outside of the lab. I appreciate your expert advice and mentoring in the lab. Your friendship, caring personality and infectious laughter has really mattered to me these years. To my co-supervisors Atle Noralf Larsen and Yvonne Pietrowski, thank you for giving me the opportunity to contribute to your exciting project. Your belief in me and the project was a huge game changer and motivation boost just when I needed it the most. Thank you for your excellent supervision throughout the experiments. I also appreciate your easy-going- and caring personalities and the patience and support you have shown. I am also sincerely thankful for the opportunity to work on the ligase project under the guidance of my co-supervisor Adele Williamson. Thank you for sharing your knowledge, creativity and your many ideas. You are truly inspiring and a great mentor. Your positive attitude and friendship have also contributed greatly to the nice atmosphere at work. The time and effort you have donated in guiding me through the writing of this thesis, from the other side of the globe, is impressive and highly appreciated. To all my supervisors – I owe you lots of gratitude for valuable input and expert advice towards finalizing this thesis. You have taught me to be a better scientist and it has been a pleasure to work with you all. Previous Head of Department Ronny Helland and previous Director of NorStruct Arne Smalås have also provided great support, thank you. I would like to thank present and former engineers Eva Bjørkeng, Trine Carlsen and Stefan Hauglid for the technical help and practical assistance with the never-ending rounds of protein purifications. Lots of gratitude also goes to Bjarte Aarmo Lund and Marcin Pierechod for valuable advising in my attempts to gain data from Microscale thermophoresis and Biacore SPR analysis. To all employees at Norstruct, for creating such a pleasant working environment, with parties, laughs, meaningless conversations, camaraderie, and scientific discussions. You all made it easy to go to work every morning, even when I felt science plotted against me. My office mates have changed throughout the years, but I have been so lucky to share office with Miriam from the beginning. I appreciate how you have been there for me every day, good or bad, and our nights out on laser light-covered dance floors have been awesome. My most recent office mate, Aili, has also been of great support. To other Norstruct friends, past and present, for supporting me through the joys and hardships both in life and during the course of my studies. I have shared so many laughs with you! I love that you “voluntarily” embraced my crazy ideas. The Norstruct Spice Girls rocks! Additional thanks go to Man Kumari, Susann and Eva for listening, for keeping me sane and for simply being there. I would also like to highlight the smiling face I met by the coffee machine every morning; thank you Kåre-Olav for continuous encouragements, your positive attitude and excellent leading on the Christmas dance floor. Staying active in this process has been important for my PhD survival. So, thank you Susann and Saana for nerdy running conversations and for happily joining me on treadmill breaks. Thank you, Erik, for fun and mindful climbing breaks, moral support and for always being so positive. To my new colleagues at the awesome genetics group at NFH, thank you for your patience when I have been a bit absent-minded this first year. Especially my office girls and my group leader, Kim Præbel, have been of great support. To Hege Devold - I appreciate having a close friend upstairs, especially one with a laughter loud enough to remind me about ongoing coffee breaks. To my great group of friends here in Tromsø, both those close to me since we first met as students about a hundred years ago, and those I got to know more recently – you know who you are and I appreciate each one of you. You bring joy and laughter to my life, with skiing, parties, luxury festival camps, dinners, training, singing (!), random coffee chats and more. Thank you for support, advice and for patiently listening to my frustrations. I look forward to spending more time with you all and contribute more socially. Gratitude also goes to my long-distance friends; Lena, Silvie and Vroni. Thank you for reaching out to me when I was distant in many ways. Our chats have meant more than you know. Most importantly, I wish to express my deepest appreciation to my family. To my parents, I would not be who I am today without your love, encouragement, and backing. You have always done everything in your power to make sure that I can achieve my dreams. I am also thankful for the unwavering support and joy from my sister, my brother in law, my nephew and niece. Dear Terje; your love, patience, support and understanding for what a PhD life is about, have meant the world to me and this thesis could not possible be completed without you. You mean the world to me. To my kids, Sigrid and Malvin, to show you anything is possible. You are an endless source of joy and remind me on a daily basis that the most important part of life is not scientific. I love you so much! Table of Contents Abstract .................................................................................................................................... viii List of papers ............................................................................................................................... ix Abbreviations and acronyms.......................................................................................................... x 1 Introduction ......................................................................................................................... 1 1.1 Nucleic acids – DNA and RNA .......................................................................................... 1 1.2 Nucleic acids and proteins – a complex on and off relationship ......................................... 4 1.2.1 Nucleic acid interacting proteins in prokaryotes........................................................ 4 1.2.2 Gene regulation ...................................................................................................... 9 1.2.3 Transcription factors and DNA recognition ............................................................. 10 1.2.4 Comparing three essential types of nucleic acid binding proteins; a transcription factor, a nuclease and a ligase ............................................................................................. 11 1.3 DNA binding by the Ferric uptake regulator (Fur)............................................................ 17 1.3.1 Iron homeostasis in bacteria .................................................................................. 17 1.3.2 Ferric uptake regulator - a global regulator involved in pathogenesis ....................... 18 1.3.3 Fur mechanism ..................................................................................................... 19 1.3.4 Fur structure......................................................................................................... 21 1.3.5 The Fur regulon (Fur-box) ...................................................................................... 22 1.3.6 Characterization of Fur from the fish pathogen Aliivibrio salmonicida ...................... 25 1.4 The role of oligoribonucleases (Orn) in mRNA decay....................................................... 26 1.4.1 mRNA metabolism in prokaryotes .......................................................................... 26 1.4.2 Oligoribonucleases degrade small RNAs in prokaryotes ........................................... 29 1.4.3 Roles of Orn