Transcriptional Elongation Roadblocking by the Lambda Bacteriophage CI Protein Open Access

Hendrickson, Cristin (Spring 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/2514nk502?locale=en
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Abstract

Transcription is a crucial cellular process that must be regulated to ensure proper gene expression. To gain a more complete picture of the control mechanisms, this experiment studies the effect of the lambda bacteriophage CI protein as a roadblock to transcriptional elongation.  Using Atomic Force Microscopy, images were taken of DNA, RNAP and the lambda repressor with nanometer resolution. Based on the location of the RNAP and the roadblocks, the effect of CI on transcriptional elongation was then determined. An initial distribution of these positions showed that CI oligomers on unlooped molecules are not effective roadblocks for RNAP while CI oligomers that mediated loops block elongation. Specifically for CI, binding at the near operator, with respect to the promoter, occurred on average 389 bp away from the start of the DNA with a standard deviation of 22 and binding at the far operator occurred on average 879 bp away from the start of the DNA with a standard deviation of 32. Statistically, on unlooped molecules RNAP was found an average of 390 bp away from the start of the DNA with a standard deviation of 246. On looped molecules, RNAP was found an average of only 214 bp away from the start of the DNA with a standard deviation of 118. This difference initially suggested the topology of the DNA-protein complex determined to what extent a CI in the path of an elongating RNA polymerase interfered with transcriptional elongation. However, only 23 looped molecules were analyzed in contrast to the 46 unlooped molecules that were used. The categorization data for CI binding and topology then showed that transcription interferes with looping by reducing the percentage of looped molecules from 55 percent in the control to 6 percent in the samples with regulated transcription. Additionally, transcription increased the percentage of molecules without any bound CI oligomers from 15 percent in the control to 64 percent in samples with regulated transcription. This additional analysis then suggests that lambda oligomers are not significant obstacles for transcription which in fact disrupts them. In that case, for the lambda repressor to maintain control of the lytic/lysogenic switch, genes nearby might be organized to diverge, so that elongating polymerase do not disturb lambda repressor oligomers holding the switch.

Table of Contents

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

 

a. Transcription as a Key to Life.…………………………………………………..................…1

 

b. Lambda Bacteriophage as a Model for Transcription……………………….......……..2

 

c. Motivation for the Study of the Looping of Lambda Bacteriophage……………...8

 

II. Atomic Force Microscopy……………………………………………………………..............…...8

 

a. Background………………………………………………………………………...........................8

 

b. Mechanics …………………………………………………………………….........................….10

 

c. Imaging……………………………………………………………………….........................…..14

 

d. Advantages and Disadvantages…………………………………………………...............18

 

III. Materials and Methods………………………………………………………………….............19

 

a. DNA for Scanning Force Microscopy…………………………………………...............19

 

b. Sample Preparation for Scanning Force Microscopy…………………………….....19

 

c. Scanning Force Microscopy and Tracing DNA Contours……………………….....22

 

IV. Results and Analysis…………………………………………………………………….............24

 

a. Control Imaging Shows Proper CI Binding……………………………………..........24

 

b. TEC Imaging Verifies RNAP Elongation……………………………………….............25

 

c. REC Imaging Initially Establishes CI Looping as an Effective Roadblock..27

 

V. Discussion……………………………………………………………………………….................30

 

a. AFM Allows Direct Visualization of Roadblocks and Transcription……….....30

 

b. CI Mediated Loops Regulate Gene Expression…………………………………........30

 

VI. Conclusion……………………………………………………………………………….................32

 

VII. References………………………………………………………………………………................32

 

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