RNA Polymerase Hybrid Passage through Roadblocks Open Access

Cartee, Allison (Spring 2023)

Permanent URL: https://etd.library.emory.edu/concern/etds/vm40xs95g?locale=en


The synthesis of messenger ribonucleic acid (mRNA) from deoxynucleic acid (DNA) by RNA polymerase (RNAP) decodes genetic information. Motor enzyme RNAP translocates at up to 50 base pairs per second in vivo, but forces and transcription factors can modulate activity and pausing, both critical for regulation. Previous studies suggest RNAP may backtrack after intrinsic pausing, which may prolong the inactive state, upon encountering a physical obstacle bound to DNA. Yet, the mechanism by which RNAP overcomes these roadblocks to ensure genetic expression is poorly understood. It is uncertain whether RNAP actively disperses a roadblock, or passively waits for its dissociation. We collected E. Coli RNAP pause times at roadblocks LacI bound at sites O1, O2, and Os, in decreasing affinity, then EcoR1. Pauses were measured as a function of forces opposing or assisting RNAP translocation via magnetic tweezers and in the presence of GreA, a protein that rescues backtracked RNAPs by cleaving nascent mRNA backed up into RNAP’s catalytic site. Regardless of magnitude, forces opposing RNAP at LacI obstacles increased average pause durations compared to assisting forces without GreA. Including GreA eliminated this increase in pause time for LacI-O1 and LacI-O2. We speculate opposing forces may promote backtracking since GreA decreased opposing force average pause times to an assisting force baseline independent of GreA. Thus, backtracking may not be critical for RNAP to overcome relatively weaker obstacles. Though LacI-Os demonstrated a similar assisting force baseline independent of GreA, adding GreA to opposing force conditions lowered average pause times beneath the assisting force baseline. Repetitive cycles of backtrack and recovery may help RNAP overcome relatively stronger obstacles. To control for any LacI-RNAP co-immunoprecipitation, we performed experiments with an inactive form of EcoR1 since this enzyme is not known to interact with RNAP. We observed similar read-through proportions as with LacI-Os. We propose that RNAP may use two different transit paths to overcome roadblocks of different relative strengths: an active pathway to dislodge stronger proteins and a passive one to wait for the dissociation of weaker proteins. Our biomechanical measurements elucidate how forces on the genome may affect RNAP behavior at roadblocks.

Table of Contents

1    Chapter 1: Introduction

1.1     Research Motivation

1.2     Nucleic Acid Structure and Mechanics

1.3     RNA Polymerase and Transcription

1.4     LacI and EcoR1 (Gln111) Roadblock Structure and Mechanics

1.5     GreA Purpose

1.6     Single Molecule Approaches

2    Chapter 2: Magnetic Tweezers (MT)

2.1     The MT Technique

2.2     MT Experimental Constructs

2.3     RNAP Visualization and Altering Transcriptional Force

2.4     Experimental Procedure

2.5     Advantages and Disadvantages to MT Analysis

3    Chapter 3: Materials and Methods

3.1     DNA Tether Construction Approach

3.2     DNA Tether Construction with PCR and Purification

3.3     Chamber Preparation for Magnetic Tweezers

3.4     Chamber Preparation for LacI

3.5     Chamber Preparation for EcoR1 Under High Salt Conditions

3.6     Magnetic Tweezer Field of Capture

3.7     Magnetic Tweezer Force Application

3.8     Observed Pause Times as Individual Histograms

4     Chapter 4: Results and Discussion

4.1     Comparing RNAP Transit for EcoR1 and LacI-Os

4.2     Pause Time Comparison between LacI and EcoR1 Roadblocks

4.3     Proposing a Hybrid Model

4.4     Future Directions

5     Chapter 5: Acknowledgements

6     Chapter 6: References 

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