Supercoiling and Protein-mediated loops in Bacterial Transcription Pubblico

Abstract

Gene regulation comprises a complex network of events. Transcription is the first step of gene expression and is regulated by proteins that bind to DNA. Such proteins may facilitate or interfere with any of the three steps of transcription: (i) initiation, (ii) elongation, (iii) termination. DNA-bound proteins may also be roadblocks along the DNA template affecting RNA polymerase (RNAP) processivity. Protein mediated looping is a ubiquitous mechanism for DNA compaction and long-range interaction between distant DNA sites, either of which can regulate transcription. Similarly, DNA supercoiling, the level of which is modulated by DNA-binding proteins, DNA-processing enzymes, including RNAP, is an inherent regulatory mechanism. To understand the interplay between transcription, protein-mediated DNA looping and supercoiling is important to understand gene regulation. The experiments described here, which leveraged the power of magnetic tweezers and tethered particle motion microscopy, and used the lac repressor protein (LacI) as a model DNA-binding and looping protein, provide insight into this problem. In particular, they show that (i) negative supercoiling can make protein-mediated looping deterministic and ergodic within the bacterial doubling time, (ii) although the LacI repressor mediating a loop is a very strong roadblock for an incoming RNAP external to the loop, (iii) RNAP may become trapped inside a loop, in which case (iv) positive supercoiling generated by RNAP progress facilitates LacI repressor dissociation from its binding site, allowing RNAP to exit from a loop. 

Table of Contents

Table of Contents

Chapter 1 Introduction. 1

§1.1 Motivation and hypothesis 2

§1.2 DNA structure. 3

§1.2.1 DNA primary structure. 3

§1.2.2 DNA structure under tension and torsion. 4

§1.2.3 Nucleoid-associated protein HU.. 7

§1.3 Transcription in Bacteria. 8

§1.3.1 Basic concepts of bacterial transcription. 8

§1.3.2 Transcription regulation: DNA looping and roadblocks 9

§1.3.3 lac repressor (LacI) and LacI mediated loop. 10

Chapter 2 Material and Methods. 11

§2.1 DNA preparation. 12

§2.1.1 DNA for the looping project in chapter 4. 13

§2.1.2 DNA for the transcription project in chapter 5. 14

§2.2 Chamber preparation. 14

§2.3 Proteins 17

§2.4 Tether particle motion (TPM) microscopy. 17

§2.4.1 Experimental setups for TPM measurements. 17

§2.4.2 TPM microscope. 18

§2.4.3 Data collection and analysis of TPM.. 19

§2.5 Magnetic tweezers (MTs) 22

§2.5.1 Experimental set-up. 22

§2.5.2 MTs microscope equipment 23

§2.5.3 Data collection and analysis of MTs. 24

§2.6 Transcription traces and pausing time measurement 27

§2.6.1 Transcription on torsional relaxed DNA and no LacI-mediated looping. 27

§2.6.2 Transcription on torsionally-constrained DNA and no LacI-mediated looping. 28

§2.6.3 Transcription on torsional relaxed DNA with LacI-mediated looping. 30

Chapter 3 Energetics of twisted DNA topologies. 32

§3.1 Introduction. 33

§3.2 Free energy of torsion-induced conformational changes in DNA.. 33

§3.2.1 Energies of DNA melting and of the B-Z transition. 34

§3.2.2 Energies of extended, curled, and plectonemic DNA.. 36

§3.3 Table of energy equations. 39

§3.4 Remarks. 42

§3.5 Discussion and Conclusions. 43

Chapter 4 Negative DNA supercoiling makes protein-mediated looping deterministic and ergodic within the bacterial doubling time. 46

§4.1 Introduction. 47

§4.2 Supercoiling makes protein-mediated looping deterministic. 47

§4.2.1 Looping probabilities of different tethers varies widely. 47

§4.2.2 Differences between lac repressor molecules contribute to the heterogeneity of looping. 50

§4.2.3 HU protein does not reduce variation in looping probabilities. 51

§4.2.4 DNA supercoiling reduces variation in looping probabilities. 54

§4.3 Supercoiling induced ergodicity within a biologically relevant timescale. 57

§4.4 Discussion and conclusion. 61

§4.4.1 HU protein induced supercoiling is insufficient to promote uniform looping dynamics. 61

§4.4.2 DNA supercoiling may lower the energy barrier of looping by juxtaposing operators through 1-D diffusion 63

§4.4.3 Conclusion. 65

Chapter 5 Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops 66

§5.1 Introduction. 67

§5.2 RNAP pauses longer at entry to than exit from LacI-loops. 67

§5.2.1 Monitoring elongation through LacI-mediated loops with tethered particle motion. 68

§5.2.2 RNAP pauses longer at entry to than exit from LacI-loops. 71

§5.3 Supercoiling plays a key role in regulating RNAP elongates through loop. 73

§5.3.1 Transcription of looped segments is slower 73

§5.3.2 RNAP surpasses LacI obstacles faster on positively supercoiled templates. 75

§5.4 Discussion and conclusion. 78

Conclusion. 81

References. 83

List of figures

Chapter 1 Introduction

Figure 1. 1. A schematic illustration of the interplay between RNA polymerase elongation, topological DNA structures, and regulation by representative proteins and enzymes. 3

Figure 1. 2. Sketch of basic structures of DNA.. 4

Figure 1. 3. Sketches of the plectoneme formation. 6

Figure 1. 4. Protein HU (blue in middle) binds with DNA (gray). (From Protein Data Bank in Europe). 7

Figure 1. 5. Cartoon of elongation by RNA polymerase. 8

Figure 1. 6. Cartoons of LacI and its mechanism of gene expression regulation. 10

Chapter 2 Material and Methods

Figure 2. 1. Plasmids, primers, and templates for the looping project in chapter 4. 13

Figure 2. 2. Plasmids, primers, and templates for transcription project in chapter 5. 14

Figure 2. 3. Image of a typical micro chamber 15

Figure 2. 4. Cartoons of the TPM experimental setup. 17

Figure 2. 5. Photograph of the TPM microscope in the Finzi-Dunlap lab used for the measurements described in this dissertation. 19

Figure 2. 6. Looping probability calculation. 20

Figure 2. 7. Looping probability vs LacI concentration. 21

Figure 2. 8. Calibration of tether length and rho square. 21

Figure 2. 9. Sketches of TPM mechanism and experimental setup. 22

Figure 2. 10. Photograph of the magnetic tweezer microscope in the Finzi-Dunlap lab used in the experiments reported in this dissertation. 24

Figure 2. 11. Tether extension calibration. 25

Figure 2. 12. Force vs magnet height curve and “hat” curve. 26

Figure 2. 13. Representative transcription traces on torsionally relaxed DNA with no loop formation. 27

Figure 2. 14. Measurement of pause time on supercoiled DNA.. 29

Figure 2. 15. An illustrative example which assembles segments from several traces that transcribing with loop formation 30

 

Chapter 3 Energetics of twisted DNA topologies

Figure 3. 1. Sketches of the twin-domain model 34

Figure 3. 2. Schematic representation of the interplay between torsion-generating DNA transactions, like transcription, conformational and topological changes in DNA, and protein binding. 43

 

Chapter 4 Negative DNA supercoiling makes protein-mediated looping deterministic and ergodic within the bacterial doubling time

Figure 4. 1. The looping probabilities of different DNA tethers vary widely. 49

Figure 4. 2. Looping percentages are uncorrelated before and after LacI replacement 51

Figure 4. 3. HU did not reduce the variation of looping probabilities amongst DNA tethers. 53

Figure 4. 4. Supercoiling dramatically reduces the variation of looping probabilities amongst DNA tethers. 56

Figure 4. 5. Long TPM recordings. 57

Figure 4. 6. Minimum observation times for ergodicity. 58

Figure 4. 7. Sufficiently long observations are ergodic. 60

Figure 4. 8. HU binding significantly contracts but mildly unwinds DNA.. 62

Figure 4. 9. HU does not dissociate under a wide range of supercoiling levels. 63

Figure 4. 10. Energy landscapes for loop closure by LacI in different conditions of tension and torsion. 65

Chapter 5 Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops

Figure 5. 1. RNAP can transcribe through a LacI-mediated loop after a pause. 68

Figure 5. 2. The LacI-mediated loop enhances and attenuates RNAP pausing at the proximal and distal binding sites respectively. 72

Figure 5. 3. RNAP transcribes a loop more slowly. 75

Figure 5. 4. Comparison of RNAP pause times at O1 with and without positive supercoiling. 77

About this Dissertation

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School	Laney Graduate School
Department	Physics
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Physics, Molecular
Parola chiave	DNA supercoiling TPM and MTs Bacterial Transcription looping
Committee Chair / Thesis Advisor	Finzi, Laura, Emory University
Committee Members	Warncke, Kurt, Emory University Burton, Justin, Emory University Artsimovitch, Irina, Ohio State University Kim, Minsu, Emory University Dunlap, David, Emory University Storici, Francesca, Georgia Tech

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