Transcription factors and supercoiling establish DNA topology that influences transcription Public
Yan Yan (Spring 2018)
Published
Abstract
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.
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 between DNA loops……………………………..…61
§ 4.2.2 Alternating loops give loop interference……………………………………………..61
§ 4.2.3 Nested loops give loop assistance……………………………………………………….64
§ 4.2.4 Side-by-side loops do not interact……………………………………………………….64
§ 4.2.5 Model to quantitate loop interactions…………………………………………………65
§ 4.3 Discussion………………………..…………………………………………………………………………….….66
Chapter 5 Protein-mediated looping of DNA under slight tension requires supercoiling ……………………………………………………………………………………….69
§ 5.1 Introduction…………………………………………………………………………………………………..…….69
§ 5.2 Results…………………………………………………………………………………………………………………72
§ 5.2.1 HU decreased tether lengths and promoted LacI-mediated looping…..…72
§ 5.2.2 Supercoiling decreased tether lengths and induced LacI-mediated looping…………………………………………………………………………………………………………………………76
§ 5.3 Discussion and conclusion……………………………………………………………………………………80
§ 5.3.1 Contraction, especially with writhe, enhances loop formation………………80
§ 5.3.2 Supercoiling lowers the free energy of DNA looping in a protein-dependent manner………………………………………………………………………………………………………82
§ 5.3.3 Comparing the effects of HU and supercoiling on DNA looping via Jloop factor……………………………………………………………………………………………………………………………83
§ 5.4 Conclusion……………………………………………………………………………………………………………84
Chapter 6 Protein-mediated loops in supercoiled DNA create large topological domain………………………………………………………………………………..86
§ 6.1 Introduction…………………………………………………………………………………………………..…….86
§ 6.2 Results………………………………………………………………………………………………………………...88
§ 6.2.1 Loop-securing LacI resists torsional stress in dynamically supercoiled DNA….………………………………………………………………………………………………………………………….88
§ 6.2.2 LacI-mediated loops preferentially trap negative supercoiling at higher tension…………………………………………………………………………………………………………………………91
§ 6.2.3 LacI-mediated loops dynamically trap large topological domains in supercoiled DNA…………………………………………………………………………………………………………..93
§ 6.2.4 Exclusion of artifacts……………………………………………………………………………..95
§ 6.3 Discussion……………………………………………………………………………………………………………97
Chapter 7 RNA polymerase pauses at lac repressor obstacles ………………………………………………………………………………………………………………..99
§ 7.1 Introduction…………………………………………………………………………………………………………99
§ 7.2 Results……………………………………………………………………………………………………………….101
§ 7.2.1 RNA polymerase overcomes pausing in magnetic tweezer experiments……………………………………………………………………………………………………………….101
§ 7.2.2 RNA polymerase may shuttling backward once encounter obstacle……104
§ 7.2.3 DNA extension can reach an extension beyond the end of DNA template……………………………………………………………………………………………………………..……..106
§ 7.3 Discussion and Conclusion…………………………………………………………………………………107
References……………………………………………………………………………………………………………109
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