Single Molecule Characterization RNA Polymerase I: Technique, Instrumentation and Experimental Development 公开

Ucuncuoglu, Suleyman (2014)

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Transcription is the prerequisite of protein synthesis. Indeed, as the factory for proteins, the ribosome is predominantly built by rRNA. In eukaryotes, this particular RNA is transcribed from rDNA genes by RNA Polymerase I (Pol I). Therefore, transcription of rDNA is vital for cellular growth and biogenesis. Pol I should produce sufficient amounts of rRNA to fulfill the increasing demand for ribosomes in the cell. The activity of Pol I is regulated up to four fold during the cell cycle, but in all, Pol I accomplishes more than 60% of the all transcription in the cell. Although Pol I is responsible for the majority of the transcriptional activity in the cell, the information related to its kinetics and mechanism is limited. This dissertation focuses on the development and use of an experimental strategy to characterize the dynamic properties of Pol I elongation. In particular, the first single molecule assay was established for monitoring Pol I elongation, and multiplexing tethered particle motion (TPM) measurements were succeeded in to simultaneously monitor hundreds of single molecules. This technical improvement was dictated by the low yield of elongating Pol I complexes. When compared to the conventional TPM setup, the throughput of the experiments was increased up to 10 fold. This new TPM setup, Pol I elongation rate, and possible pause locations along the rDNA template were measured. The average elongation rate measured was 20 nt/s at 200 µM NTP and the pause free rate was around 50 nt/s which is comparable with the estimated in vivo rate. Furthermore, a frequent pause location was identified around 200 bp downstream of the promoter. This finding is in line with the recent finding of a prominent pause location in prokaryotic cells.

In conclusion, the TPM optical and data collection/storage setup was optimized. This system was used to directly measure the elongation rate of Pol I and its pause probability along the template. These dynamic characteristics provide important insight into the mechanism of Pol I transcriptional elongation which may be valuable in engineering anticancer drugs aimed at stopping cell proliferation by interfering with ribosome assembly.

Table of Contents

Chapter 1 1

Introduction 1

1.1 The Tethered Particle Motion technique (Tethered Particle Microscopy) 2

1.1.1 TPM Experimental Setup 3

1.1.2 Experimental protocol for TPM experiments 4

1.1.3 TPM Measurements 5

1.2 RNA Polymerase 7

1.2.1 Types of RNA Polymerases 8

1.2.2 Transcription by RNA Polymerase I 9

1.2.3 Structure of RNA Polymerase I 10

1.3 Motivation and Hypothesis 11

Chapter 2 14

Calibration of Tethered Particle Motion 14

2.1 Introduction 15

2.2 Materials and Method 16

2.2.1 DNA Preparation 16

2.2.2 Chamber Preparation 18

2.2.3 Particle tracking, Data acquisition, and Instrumentation 19

2.2.4 Data Preprocessing and Drift Calculations 21

2.2.5 Symmetry Test 21

2.3 Results 23

2.3.1 Mean Square Excursion 24

2.4 Conclusion 27

Chapter 3 28

Multiplexed Tethered Particle Motion 29

3.1 Introduction 29

3.2 Materials and Methods 30

3.2.1 Sample Preparation for Transcription experiments 30

3.2.2 Sample Preparation for Gyrase wrapping experiments 32

3.2.3 Chamber assembly 32

3.2.4 Optical Setup 34

3.2.5 Hardware and Software 35

3.2.6 Drift correction and bead selection 37

3.3 Results and Discussion 39

3.3.1 Accuracy of radial symmetry tracking 39

3.3.2 Effect of Averaging Time 42

3.3.3 Comparison of DIC and Dark Field Microscopy 44

3.3.4 Gyrase Wrapping Experiments 47

3.3.5 Effects of Compression on the Movie file 48

3.4 Discussion 51

Chapter 4 52

Single Molecule Measurements of RNA Polymerase I Elongation 52

4.1 Background 53

4.2 Materials and Methods 55

4.2.1 Fabrication of TPM microchambers 55

4.2.2 Preparation of Transcription reagents 56

4.2.3 Sample Preparation 61

4.2.4 TPM Measurements of stalled and elongating Pol I complexes 62

4.2.5 Simulations 62

4.3 Results and Discussion 64

4.3.1 Elongation Rate 66

4.3.2 Processivity RNA Polymerase I 69

4.3.3 Pause sites on rDNA 73

4.3.4 Random Walk Hypothesis 76

4.3.5 Energy Barriers of Elongation 78

4.3.6 Kinetic rate of transcription 79

Chapter 5 83

Conclusions 83

5.1 Prerequisite Experiments: Calibration of TPM Data 84

5.2 A Must for Low Yield of Transcriptional events: Multiplexed TPM 85

5.3 Pol I processivity 86

5.4 Pol I elongation rate and pausing 88

References 92

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