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Studies on transcription in escherichia coli

Sendy, Bandar (2017)
Ph.D. thesis, University of Birmingham.

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Abstract

The expression of genes is tightly controlled, predominantly at the point of transcription. RNA polymerase (RNAP) must first bind to a deoxyribonucleic acid (DNA) promoter upstream of a gene to transcribe it. However, the ability of RNAP binding is dictated by the core promoter DNA sequence, the presence of transcription activator or repressor proteins and numerous other factors. The strength of promoters has been indirectly measured. Only a few studies have attempted to directly address the RNAP flux through transcription units, and further studies are still required.
In the current study, the aim was to directly correlate RNAP gene transcription with the strength of core promoter elements. To do that, I employed the direct method of chromatin immunoprecipitation (ChIP), followed by quantification of immunoprecipitated DNA. For promoter regions, this method directly measures the occupancy by RNAP; for regions within transcription units, the flux of the RNAP was deduced.
A range of semisynthetic promoters, with different combinations of core promoter elements to obtain different levels of expression, was used to validate our method. This direct method enabled the calculation of “promoter competitivity”, “promoter occupancy index” (POI), RNAP “escape index” (EI), “fragment occupancy percentage” (FOP) and the time interval between transcribing RNAPs (Tint). On the basis of Tint, the number of RNAPs crossing any DNA sequence of interest per second (polymerase per second; PoPS) was calculated. Surprisingly, the results of the present study revealed that the RNAPs are well separated during transcription of the \(lac\) operon.

Type of Work:Ph.D. thesis.
Supervisor(s):Busby, Steve
School/Faculty:Colleges (2008 onwards) > College of Life & Environmental Sciences
Department:School of Biosciences, Institute of Microbiology & Infection
Subjects:QH426 Genetics
QR Microbiology
Institution:University of Birmingham
ID Code:7576
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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