Design of an experimental strategy to study the effect of rNTPs on transcriptional elongation by E. Coli RNA Polymerase Open Access

Abstract

Due to the high concentration of ribonucleotides (rNTPs) versus lower concentration of deoxy-ribonucleotides (dNTPs) in the cytoplasm, misincorporation of rNTPs into a DNA strand during replication is likely to occur. This strand of DNA may then participate in transcription by RNA Polymerase. The misincorporated rNTPs may serve as a transcriptional roadblock by affecting an RNA Polymerase as it transcribes the DNA strand. We hypothesize that rNTPs may act as an obstacle to transcription, slowing or even halting RNA Polymerase progress as it reads the misincorporated template. We used Protein Induced Fluorescent Enhancement (PIFE) to study the effects of rNTPs on RNA Polymerase by strategically placing a Cy3 fluorophore on the DNA strand. To analyze PIFE signals, an objective-type total internal reflection microscope with a 532 nm laser was used. Using a control strand—with no rNTPs—optimal conditions for the experiment were discovered. Due to non-specific interactions between RNA Polymerase and the DNA template, excessive protein concentration lead to spurious PIFE signals. Conversely, concentration that we too low prevented detection. We determined that an RNA Polymerase concentration of 0.05 U/uL was an optimal working condition. By comparing PIFE activity of different DNA constructs we found that RNA Polymerase enhances Cy3 fluorescence when placed within 67 bp of the dye; yet, when placed 245 bp away little fluorescence enhancement is observed. Together, these findings will help us to experimentally determine whether or not RNA Polymerase will behave as a roadblock during transcription and slow RNA Polymerase’s progress down the strand. When observing PIFE in future experiments, we know that the RNA Polymerase less than 245 bp away and at least 67 bp away from the rNTPs and Cy3 fluorophore. 

Table of Contents

I............. Introduction...................................................................................................................... 1

Structural Differences between DNA and RNA Molecules.......................................................... 1

A.           Cellular Replication and Transcription...................................................................... 2

Cytoplasmic Nucleotides and Misincorporated Ribonucleotides............................................... 2

Transcription of Misincorporated Ribonucleotides by RNA Polymerase.................................... 3

II............ Experimental Design......................................................................................................... 4

Fluorescence................................................................................................................................ 4

A.           Cy3 Fluorophore........................................................................................................ 5

B.           Protein-Induced Fluorescence Enhancement........................................................... 6

Total Internal Reflection Fluorescence Microscope.................................................................... 7

A.           TIRF with Cy3 Fluorophores...................................................................................... 9

B.           Preparation of the Experimental Chamber............................................................... 9

C.            Flowing in DNA into the Chamber........................................................................... 10

D.           Photobleaching........................................................................................................ 11

E.            Data Readout from CCD Camera............................................................................. 12

III........... Initial DNA Construct...................................................................................................... 14

Annealing Fragment 2................................................................................................................ 14

Polymerase Chain Reactions of Fragment 1 and Fragment 3................................................... 15

Combining Fragment 1 with Fragment 2 and Fragment 3........................................................ 16

A.           Restriction Digestion............................................................................................... 16

B.           Ligation Reaction..................................................................................................... 18

Gel Electrophoresis.................................................................................................................... 20

A.           Agarose Gel............................................................................................................. 20

B.           Polyacrylamide Gel.................................................................................................. 21

IV.......... Results and Analysis........................................................................................................ 22

Data with the Initial DNA Construct.......................................................................................... 22

Synthesizing the DNA Molecule using Initial DNA Design......................................................... 31

V........... Reliability of RNA Polymerase Affecting Cy3 Fluorophore............................................. 38

Design of DNA Constructs.......................................................................................................... 38

Experimental Results and Analysis............................................................................................ 40

VI.......... Conclusion....................................................................................................................... 51

VII......... References...................................................................................................................... 52

About this Honors Thesis

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School	Emory College
Department	Physics
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biophysics, General
Keyword	PIFE Total Internal Reflection Fluorescence Fluorophore TIRF Transcriptional Roadblock Protein Induced Fluorescence Enhancement Total Internal Reflection Microscope Transcription RNA Polymerase Cy3
Committee Chair / Thesis Advisor	Finzi, Laura, Emory University
Committee Members	Orloff, Gregg, Emory University Dunlap, David, Emory University Seitaridou, Effrosyni, Emory University

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