An Experimental Framework to Examine the Influence of Promoter Architecture and Genomic Context on Gene Expression
Transcription is a fundamental process of gene expression. Information stored in DNA is transcribed into different types of mobile RNA, which play a role in various essential processes of the cell, e.g. translation. However, cells do not need all the information stored in their DNA at the same time....
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|Summary:||Transcription is a fundamental process of gene expression. Information stored in DNA is transcribed into different types of mobile RNA, which play a role in various essential processes of the cell, e.g. translation. However, cells do not need all the information stored in their DNA at the same time. Therefore, the process of transcription gets regulated by a plethora of mechanisms. One frequently discussed but poorly understood mechanism of transcription regulation is DNA supercoiling [Travers and Muskhelishvili, 2005]. Whereby, the process of transcription itself affects the DNA-topology up- and downstream of the transcription machinery as described in the twin supercoiling domain model [Liu and Wang, 1987]. This phenomenon is called Transcription Coupled DNA Supercoiling (TCDS). It has also been shown that genes react individually to changes in DNA supercoiling and that there is a selection pressure on adapting to the DNA supercoiling levels emitted by neighbouring gene expression [Sobetzko, 2016]. The system in which promoters react to changes in DNA supercoiling is as diverse as there are promoters; notably, some promoters seem not to respond to DNA supercoiling at all. Thus, this raises the question as to which elements within different promoter types cause them to respond to TCDS so differently. In this thesis, I built a pipeline to investigate the effects of TCDS and DNA supercoiling on promoters. Firstly, I created a plasmid toolbox, which allows modular assembly of transcription units. The central feature of this toolbox is the flexibility to test different arrangements of multiple transcription units. I achieved this by adapting the well established Modular Cloning (MoClo) standard [Weber et al., 2011] and build my toolbox around it. I thus created a system that works on both its own and is compatible with the existing standard MoClo protocol.
In the second part of this thesis, I established an experimental pipeline using synthetic σ70-promoters to investigate the influence of DNA supercoiling on transcription. The experimental setup allowed precise changes in parts of the promoter and at the same time created a library of these promoters. Using this pipeline to investigate the spacer region of the promoter, I was able to confirm that the spacer influences the promoter strength. Further, I showed that the promoter spacer has only a limited effect on the supercoiling sensitivity of a promoter. I also showed that a 5‘-TGTG-3‘ motif in the spacer region could lower transcription by enhancing RNA-polymerase (RNAP)-binding. Moreover, the experimental setup also showed the constraints of using the DNA-relaxing drug novobiocin on a plasmid-based system. Hence, to further investigate the effects of TCDS on neighbouring transcription, I applied an optogenetically-controllable promoter to the previously established pipeline. Finally, I began to explore the possibility of integrating my experimental promoter setup into any genomic position. As such, a CRISPR/Cas9-based homologous re-combination system was developed further to make it modular and compatible with the Modular Cloning protocol. I could show the first features of this system to work.|
|Physical Description:||149 Pages|