Genetic Studies of the Atpase Uup and the Ribosomal Protein

Elucidating ribosomes-genetic studies of the ATPase Uup and the ribosomal protein L1 by Katharyn L Cochrane A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular, Cellular, and Developmental Biology) in the University of Michigan 2015 Doctoral Committee: Professor Janine Maddock, Chair Associate Professor Matthew Chapman Associate Professor Lyle Simmons Professor Nils Walter Dedicated to: My parents, without whom I would never have embarked on this journey, and without whose support I would never have finished; My daughter, who has had to do without me more than she would like, and who has motivated me to press on; My family, whose love and support I treasure; And those dear friends whose prayers and encouragement carried me on this journey. ii Acknowledgments I would like to begin by thanking my PhD advisor, Dr. Janine Maddock, for all her support, both personally and professionally. Janine has not only mentored me as a scientist, but has also been a powerful encourager and advocate for me as I ventured to make personal choices and navigated the consequences of those choices. Without her care and flexibility, I would not have continued in my pursuit of this PhD. She has provided an example for me in the way she vehemently cares for individuals, to a degree that far exceeds the requirements of her job. Janine’s support has changed my life for the better, and I am extremely grateful. Likewise, I am indebted to Dr. Carlos Gonzalez, whose kind teaching and belief in me motivated me to dare to aspire to this path, and who first directed me to Michigan. It was in his lab I developed an interest in pursuing research science. I would not have attempted this without his confidence in me. I am also thankful to the excellent post-doctoral researchers who have taught and encouraged me while in the Maddock lab. I thank Dr. Sonia Bardy for “showing me iii the ropes” and providing a solid base in many of the techniques and lab skills I used in the course of my research. I thank Dr. Richard Zielke for his sense of humor and the sense of belonging he shared with me, and for his foundational work on the topics discussed in this thesis. I thank Dr. Raji Janakiraman for the commiseration we shared, and for keeping me company during the late nights we spent in lab together working on our lab reports. I especially thank Dr. Lolita Piersimoni for her spunk, ready smile, and humor that helped make the darkest days brighter, and for her significant contributions to my professional development. She has generously shared her time in many discussions to help me develop and test my ideas as well as in lending her much more able hands in helping me to obtain many “just one more” pieces of data. Our many discussions have been invaluable in my journey to independent thinking worthy of a PhD. In many ways, she has been an older sister to me in this journey. I would also like to thank all the other Maddock lab members I have known while here, especially Lance Shultz and Dr. Billy Clifford-Nunn. Your friendship has meant a great deal to me. The members of my committee, Dr. Matt Chapman, Dr. Lyle Simmons, and Dr. Nils Walter have all been extremely helpful in my journey. Possibly consciously, probably unconsciously they have all in turn been the one smiling face to give me hope and the grim disciplinarian to require me to keep aspiring higher. I have found iv courage in hope, and am glad of what their sternness helped goad into development. I appreciate their faith in me, and their genuine good-will. I would also like to thank the members of the wider MCDB community who have touched me during my time here. In particular, I thank Dr. Mohammed Akkabounne for taking me in and teaching me neurobiology, Mary Carr for being a knowledgeable and dependable resource, my classmates, particularly those with whom I have kept in contact, for being a kind and caring support group as we all adjusted to this new venture, and members of the micro-bio “Super Group” for their many intellectual contributions to my work. A special mention goes to Dr. Marcus Ammerlaan whose passion for teaching and keen sense of humor have been refuges for me when research felt overwhelming. Finally, I would like to thank my family, particularly my parents, grandparents and siblings, for closing ranks around me during this time. Your support has been another indispensible help in completing this degree. You have all done much more than I could ask to help me succeed, and I cannot thank you enough. I also thank my daughter for gladly becoming part of Mommy’s science life, and generously sharing me with this PhD. Your smile and the knowledge that the outcome of this degree program is vitally important in your life as well as in mine have motivated me when nothing else would have mattered. v Thank you to all who have walked this road with me, to all who have lit the way, to all who have cheered me on. Thank you to all who have helped me walk another little while, and to all who have carried me when I was too weak to keep going on my own. This is your victory as well as mine. vi Table of Contents Dedication ...……………………………………………………………..………………………… ii Acknowledgements ………………………………………………………………………..….. iii List of Tables ……………………………………………………………………………………… ix List of Figures ……………………………………………………………………………………. x Abstract ……………………………………………………………………………………………. xii Chapter 1. Introduction The bacterial ribosome …………………………………………………… 1 Ribosomal protein L1 ……………………………………………………… 3 Ribosomal Protein L11 ……………………………………………………. 7 Translation ………………..……………………………………………………. 8 Initiation ……………………………………………………………….. 8 Elongation …….…………………………………………………….. 11 Termination/Recycling ……………………………………….. 12 Elongation Factors ………………………………………………………… 14 GTPase elongation protein family ……………………….. 14 EF-Tu ………………...……………………………………….. 15 EF-G ………..………………………………………………….. 18 LepA …………………………………………………..………. 20 BipA …………………………………………………………... 22 Non-GTPase bacterial elongation factors …………….. 24 vii EF-P ……………………………………………………………. 24 EttA …………………………………………..……………….. 26 References ……………………………………………………………………. 27 2. Uup is an ATPase involved in translation …………………..……… 49 Introduction …………………………………………………………………. 49 Methods ………………………………………………………………………… 54 Results ………………………………………………………………………….. 59 Discussion ……………………………………………………..……………... 70 References ………………………………………………………….………… 76 3. Characterization of a ΔrplA mutant of E. coli ……………………… 97 Introduction …………………………………………………………………. 98 Methods …………………………………………………………………….... 100 Results ………………………………………………………………………… 102 RpoS regulation/regulon ………………………………...… 105 Ribosome/translation connected suppressors …. 110 DNA replication-involved suppressors ……………… 115 Proteins of unknown relevance ………………………… 116 Discussion ……………………………………………………..……………. 118 References ………………………………………………………………….. 121 4. Future Directions …………………………………………………………….. 141 viii List of Tables Table 2.1 Strains and plasmids (used in Chapter 2) …. ………...…… 80 Table 3.1 Strains and plasmids (used in Chapter 3) …..…………... 130 Table 3.2 Functional classes of ΔL1 slow growth in the cold high copy suppressing proteins ……………………………………………………. 136 ix List of Figures Figure 1.1 Features of the large ribosomal subunit …………………. 43 Figure 1.2 Initiation of translation involves formation of a 30S pre- initiation complex and subsequent joining of the 50S subunit .. … 44 Figure 1.3 EF-G catalyzed translocation …………………………………. 45 Figure 1.4 Release Factors 1 and 2 interact with the stop codon in the A-site of the ribosome ………………………………………………………. 46 Figure 1.5 Domain structure of the elongation GTPase family …. 47 Figure 1.6 E-site translation factors ……………………………………….. 48 Figure 2.1 Uup is a ∆bipA suppressor …………………………………….. 82 Figure 2.2 Growth consequences of plasmid-borne mutant Uup in presence and absence of endogenous Uup ………………………………. 83 Figure 2.3 Growth consequences of plasmid-borne mutant Uup on strains lacking BipA ………………………………………………………………. 84 Figure 2.4 Dominant negative Uup variants associate with ribosomes more strongly than other variants ………………………….. 85 Figure 2.5 Uup is found on ribosomes in vitro and in vivo …...…... 86 x Figure 2.6 Overexpression of Uup results in earlier correction of an OD600-dependent ΔbipA 18° ribosome profile defect ………..…… 87 Figure 2.7 Uup and EF-G suppress ΔbipA rifampicin sensitivity .... 88 Figure 2.8 Translation ﬁdelity in a ∆bipA mutant …………………….. 89 Figure 2.9 Translation ﬁdelity in a ∆uup mutant ……………………... 90 Figure 2.10 Deletion of Uup results in decreased polysomes ……. 91 Figure 2.11 Uup and EttA are similar ………………………………………. 92 Figure 2.12 While mutants of uup and ettA have different phenotypes, high-copy Uup and EttA suppress each other’s ribosome profile defects………………………………………………………...... 93 Figure 2.13 Overexpression, but not loss, of Uup reduces viability………………………..…………………………………………………………. 94 Figure 2.14 Uup may be important for translation of topA ………. 95 Figure 2.15 Uup may also be an E-site translation factor…………… 96 Figure 3.1 Effects of loss of ribosomal protein L1 on P-site and A- site fidelity ………………………………………………………………………….. 132 Figure 3.2 Loss of the L1 protein results in an accumulation of 50S ribosomal subunits ……………………………………………………………… 133 Figure 3.3 High copy suppressors of ΔL1 cold sensitivity ………. 134 Figure 3.4 Relationship of RpoS-related high-copy suppressing proteins of ΔL1 slow growth in the cold ………………………...……… 138 Figure 3.5 The ribosome-binding/affecting sites of ribosome- related suppressing proteins clusters around

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