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Distribution Agreement in Presenting This Thesis Or Dissertation As a Partial Fulfillment of the Requirements for an Advanced De Distribution Agreement In presenting this thesis or dissertation as a partial fulfillment of the requirements for an advanced degree from Emory University, I hereby grant to Emory University and its agents the non-exclusive license to archive, make accessible, and display my thesis or dissertation in whole or in part in all forms of media, now or hereafter known, including display on the world wide web. I understand that I may select some access restrictions as part of the online submission of this thesis or dissertation. I retain all ownership rights to the copyright of the thesis or dissertation. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. Signature: _____________________________ ______________ Yan Yan Date Transcription factors and supercoiling establish DNA topology that influences transcription By Yan Yan Doctor of Philosophy Physics _________________________________________ Laura Finzi Advisor _________________________________________ William Kelly Committee Member _________________________________________ Harold Kim Committee Member _________________________________________ Minsu Kim Committee Member _________________________________________ Eric Weeks Committee Member Accepted: _________________________________________ Lisa A. Tedesco, Ph.D. Dean of the James T. Laney School of Graduate Studies ___________________ Date Transcription factors and supercoiling establish DNA topology that influences transcription By Yan Yan B. S., Shandong University, China, 2011 Advisor: Laura Finzi, Ph. D. An abstract of A dissertation submitted to the Faculty of the James T. Laney School of Graduate Studies of Emory University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physics 2018 Abstract Transcription factors and supercoiling establish DNA topology that influences transcription By Yan Yan Protein-mediated DNA looping is ubiquitous in chromatin organization and gene regulation both in eukaryotes and prokaryotes. It occurs when one or more protein(s) bridge two distant sites on the double helix. In principle, different protein-mediated loops may, interact giving rise to more complex regulation patterns. Using the tethered particle motion technique, the probability of looping by the lac repressor protein (LacI) was shown to depend on LacI concentration, loop size and binding affinity of operators. Furthermore, it was shown that DNA loops mediated by LacI and the λ bacteriophage repressor protein, λ CI, interact differently depending on the topological arrangement: side-by-side loops do not affect each other, nested loops assist each other’s formation, while alternating loops inhibit each other’s formation. These observations provided clear support for the loop domain model for insulation. Nucleoid associated protein (NAPs) and/or supercoiling could both be responsible for the fact that in vivo levels of LacI-mediated looping are higher than those measured in vitro for a range of large loop sizes in identical DNA templates. Using magnetic tweezers, physiological levels of negative supercoiling were shown to drive the looping probability from 0 to 100 % under slight tension that likely exists in vivo. In contrast, even saturating (micromolar) concentrations of HU couldn’t raise the looping probability above 30 % in similarly stretched DNA or 80% in DNA without tension. Furthermore, it was shown that loops that formed in supercoiled DNA create topological domains that may exceed the loop segment length (distance between protein binding sites). This is relevant to regulation by distant elements. Magnetic tweezers were also used to show that RNAP halts for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation. Puzzlingly, RNAP seems to slide back to the promoter immediately after encountering a protein and reinitiate elongation. These observations still need to be understood. Transcription factors and supercoiling establish DNA topology that influences transcription By Yan Yan B. S., Shandong University, China, 2011 Advisor: Laura Finzi, Ph. D. A dissertation submitted to the Faculty of the James T. Laney School of Graduate Studies of Emory University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physics 2018 Acknowledgement I owe my deepest gratitude to my advisor, Prof. Laura Finzi, for her generous encouragement, guidance and support throughout my graduate academic studies. These pages would be impossible without her kind mentorship and friendship over my long stay in the Physics Department. I also sincerely thank Dr. David Dunlap for the kind advising and patient teaching. I am heartily thankful to my Ph. D. advising committee members: Dr. William Kelly, Dr. Harold Kim, Dr. Minsu Kim, Dr. Eric Weeks for their encouraging words, suggestions for my research, thoughtful criticism, and for attending my annual meetings. I am grateful to my previous coworkers, Dr. Sandip Kumar and Dr. Mónica Fernández. They greatly helped me mastering a variety of lab techniques and developing critical thinking with constructive discussion during my first year of research in the lab. I would like to thank the group members, Dr. Suleyman Ucuncuoglu, Dr. Yue Ding, Dr. Diane Wiener, Dr. Daniel Kovari, Dr. Zsuzsanna Vörös, Wenxuan Xu for thoughtful discussions and for their friendship. Many thanks also to the undergraduate students who worked with me, Alex Zhang, James Boyle, Hayes Burdette-Sapp. They facilitated my work with sample preparations, experiments, and data analysis. They all did a great job. I am indebted to our collaborators Dr. Fenfei Leng, for generously supplying the HU protein and plasmids, and Dr. Kathleen Matthews for generously supplying the LacI protein. I would also like to thank my parents, my father Chengchun Yan, and my mother Chongzhen Bai. They always trust, support, and encourage me to overcome difficulties in my life. I owe much to my advisors in China, Prof. Ming Li, Prof. Chunhua Xu and their previous graduate students Dr. Zhe Pang and Dr. Shuang Wang. They are the first people who lead me into this field and taught me a lot in my early scientific research. I also thank them for their collaboration on my qualifier proposal which is not included in this dissertation. I received guidance and assistance from many people and friends over the years. It is impossible to list and thank them all. This dissertation is dedicated to all of them as well. Table of contents Chapter 1 Introduction ………………………………………………………………………………..….1 § 1.1 DNA structure……………………………………………………………………………………………………….1 § 1.1.1 DNA primary structure…………………………………………………………………………...1 § 1.1.2 DNA secondary structure and polymorphism of DNA structure………………3 § 1.1.3 DNA tertiary structure, DNA supercoiling……………………………………………….4 § 1.1.4 DNA quaternary structure……………………………………………………………………...5 § 1.2 Introduction of HU protein……………………………….…………………………………………………..6 § 1.3 Introduction of transcription…………………………………………………..……………………….……7 § 1.3.1 Central dogma of molecular biology…………………………………………………….…7 § 1.3.2 Components and function of RNA polymerase……………………………………....9 § 1.3.3 Transcription steps: initiation, elongation and termination…………………..10 § 1.3.4 Transcriptional regulation……………………………………………………………………..14 § 1.4 The lac repressor protein, LacI, and LacI-mediated DNA looping………………………….14 § 1.4.1 Lac Operon and structure of the LacI protein………………………..………………14 § 1.4.2 LacI mediated DNA looping study in vivo and in vitro…………………………….16 § 1.5 DNA topological domains………………………………………..…………………………………………..20 § 1.6 Experimental techniques………………………………………………………..……………………………23 § 1.6.1 Single molecule techniques………………………………………………………..…………23 § 1.6.2 Tethered-particle motion……………………………………………………………..……….23 § 1.6.3 Magnetic tweezers…………………………………………………………..……………………26 Chapter 2 Material and Methods…………………………………………………………..…..29 § 2.1 Preparation of DNA constructs…………………………………………………………………………....29 § 2.2 Buffer recipes………………………………………………………………………..…………………………….32 § 2.3 Microchamber Preparation…………………………………………………………………..……………..34 § 2.4 Tethered Particle Motion (TPM) experiments ………………………………………………….….35 § 2.5 Magnetic tweezers (MTs) experiments………………………………………………..………………37 § 2.6 Estimation of the HU concentration in an E. coli cell………………………………………..….41 § 2.7 Exclusion of artifacts in the study of topological domains…………………………..……….41 § 2.8 Measuring the coiling and extent of topological domains…………..……………………….43 § 2.9 Obtaining the gyre size……………………………..…………………………………………………………43 Chapter 3 protein concentration, operator affinity and loop size dependence of LacI-mediated DNA looping…………………………….………...44 § 3.1 Introduction ………………………………………………………………………………………………..…….44 § 3.2 Results…………………………………………………………………………………………….………………….47 § 3.2.1 Two looped states are observed in the experimental trace ……………..…..47 § 3.2.2 LacI concentration dependence of protein mediated DNA looping ………48 § 3.2.3 Operator affinity dependence of protein mediated DNA looping……….…50 § 3.2.4 Loop size dependence of protein mediated DNA looping ………..……………50 § 3.3 Discussion………………………………..………………………………………………………………………….52 Chapter 4 Quantitation of interactions between two DNA loops demonstrates loop domain insulation in E. coli cells ………………..…....57 § 4.1 Introduction…………………………………………………………………………………………………..…….57 § 4.2 Results………………………..…………………………………………………………………………………….…61 § 4.2.1 Quantitation of interactions
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