Transcriptional Regulation by the Bacteriophage 186 Wrapping Protein Open Access

Borjas, Gustavo (Spring 2021)

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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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