Transcriptional Regulation by the Bacteriophage 186 Wrapping Protein Open Access
Borjas, Gustavo (Spring 2021)
Abstract
DNA provides the information necessary for life. Its code contains the instructions necessary to make various types of RNA molecules during a process known as transcription. The process must be tightly regulated to express only the types of RNA needed at any particular time during the cell cycle. Transcription factors regulate transcription, many of which bind directly to DNA. The aim of this study was to further understand transcriptional regulation by what is a fundamental mode of physical regulation by proteins: DNA wrapping around a wheel-like protein complex. Thus, we used nanometer-resolution imaging by Atomic Force Microscopy to understand the effect of the model bacteriophage 186 CI repressor protein on transcriptional elongation by RNA polymerase (RNAP). In the biological context, 186 CI acts as a genetic switch between the bacteriophage lysogeny/lytic decision by inhibiting RNAP initiation when bound to promoter sites. In this study, 186 CI—which wraps DNA like the eukaryotic histone octamer—was used as a model for the regulatory wheel mode. The topographical images of DNA-protein complexes were processed with MATLAB® to obtain DNA length, protein binding position along DNA as well as protein area and height data. It was found 186 CI and RNAP could be differentiated by their height: 1.5 ± 0.3 nm and 2.5 ± 0.4 nm, respectively. Image analysis showed the wheel mode of roadblocking does not effectively stop elongation by RNAP, but rather controls the genetic switch in bacteriophage DNA via inhibition of RNAP initiation. Furthermore, the reduced 186 CI diameter on DNA in images with active transcription indicates RNAP may partially break the 186 CI wheel by knocking off wheel dimers. Since it has also been shown histone dimers are lost from nucleosomes on the elongation pathway, we speculate loss of units from regulatory protein complexes could be a general mechanism of chromatin remodeling which facilitates roadblock removal from elongation pathways.
Table of Contents
I. Introduction 3
A. DNA Transcriptional Regulation 3
B. Bacteriophage 186 as Model for Transcriptional Regulation by Wrapping 6
C. DNA Wrapping as a Common Regulatory 9
II. Principles of Atomic Force Microscopy 11
A. Scanning Modes 12
B. Advantages and Disadvantages of AFM 13
III. DNA Modification 15
A. Polymerase Chain Reaction Extracts Desired Sites 15
B. Recombinant Plasmid 18
IV. Materials and Methods 20
A. Genetic Recombination 20
B. Sample Preparation 20
C. AFM Image Scan 21
V. Image Processing and Analysis 24
A. Tracer GUI 24
B. Particle Analysis 25
C. Linking Particle and Molecule Data 29
VI. Results and Analysis 31
A. Control Imaging 31
B. Differentiating 186 CI and RNAP 33
C. Transcription Experiments 36
VII. Discussion 39
A. Transcriptional Control through Chromatin Remodeling 39
B. RNAP Breaks the Wheel 40
VIII. Conclusion 43
IX. References 44
X. Appendix 46
A. Recombinant Plasmid Sequence 46
B. Primer Sequences 48
C. MATLAB® Code 49
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