THE SPATIAL ORGANIZATION OF TRANSCRIPTION IN E. COLI

bacteria;E. coli;transcription regulation;spatial organization;gene expression;Biophysics

Weng, XiaoliSchleif, Robert F. ;

Johns Hopkins University

关键词: bacteria;    E. coli;    transcription regulation;    spatial organization;    gene expression;    Biophysics;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/59143/WENG-DISSERTATION-2018.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Transcription, the process of converting genetic information stored in DNA to RNA, lies at the heart of gene expression. Transcription has been studied extensively in vitro to probe its mechanistic detail; however, these conditions differ vastly from the complex environment inside a living cell. Spatial distributions of molecular components have been shown to be an important facet of gene regulation in living systems. First, to gain insight into the regulation of gene expression at the cellular level, we investigated the spatial distributions of various molecular components of transcription in E. coli and their physical correlation with each other using superresolution fluorescence microscopy (Chapter 2). Our results show that while dense RNAP clusters are highly colocalized with rrn operons and nascent rRNA transcripts during fast growth, these RNAP clusters are present independent of rRNA transcription activity, and are likely stably associated with the underlying chromosome structure. Second, we provided the first direct observation of transcription factor mediated DNA looping in live E. coli, and calculated in vivo looping frequencies in the context of different regulatory regimes (Chapter 3). Third, we initiated the development of a reconstituted CRISPR-Cas system to effectively visualize sequence specific genomic sites in live cells in a high-throughput manner. This system would allow for unprecedented insight into eukaryotic genome architecture in intact living cells; Chapter 4 details our efforts in optimizing the reconstituted CRISPR-Cas system first using in vitro assays.