Dissertation/ Thesis
Transcription-Coupled DNA Supercoiling in Escherichia Coli: Mechanisms and Biological Functions
| العنوان: | Transcription-Coupled DNA Supercoiling in Escherichia Coli: Mechanisms and Biological Functions |
|---|---|
| المؤلفون: | Zhi, Xiaoduo |
| المصدر: | FIU Electronic Theses and Dissertations. |
| بيانات النشر: | FIU Digital Commons. |
| المجموعة: | Florida International University |
| مصطلحات موضوعية: | Transcription-coupled DNA supercoiling, Promoter strength, E. coli topA strains, Twin-supercoiled-domain model of transcription, Hypernegatively supercoiled DNA, Biochemistry |
| الوصف: | Transcription by RNA polymerase can induce the formation of hypernegatively supercoiled DNA both in vivo and in vitro. This phenomenon has been explained by a “twin-supercoiled-domain” model of transcription where a positively supercoiled domain is generated ahead of the RNA polymerase and a negatively supercoiled domain behind it. In E. coli cells, transcription-induced topological change of chromosomal DNA is expected to actively remodel chromosomal structure and greatly influence DNA transactions such as transcription, DNA replication, and recombination. In this study, an IPTG-inducible, two-plasmid system was established to study transcription-coupled DNA supercoiling (TCDS) in E. coli topA strains. By performing topology assays, biological studies, and RT-PCR experiments, TCDS in E. coli topA strains was found to be dependent on promoter strength. Expression of a membrane-insertion protein was not needed for strong promoters, although co-transcriptional synthesis of a polypeptide may be required. More importantly, it was demonstrated that the expression of a membrane-insertion tet gene was not sufficient for the production of hypernegatively supercoiled DNA. These phenomenon can be explained by the “twin-supercoiled-domain” model of transcription where the friction force applied to E. coli RNA polymerase plays a critical role in the generation of hypernegatively supercoiled DNA. Additionally, in order to explore whether TCDS is able to greatly influence a coupled DNA transaction, such as activating a divergently-coupled promoter, an in vivo system was set up to study TCDS and its effects on the supercoiling-sensitive leu-500 promoter. The leu-500 mutation is a single A-to-G point mutation in the -10 region of the promoter controlling the leu operon, and the AT to GC mutation is expected to increase the energy barrier for the formation of a functional transcription open complex. Using luciferase assays and RT-PCR experiments, it was demonstrated that transient TCDS, “confined” within promoter regions, is responsible for activation of the coupled transcription initiation of the leu-500 promoter. Taken together, these results demonstrate that transcription is a major chromosomal remodeling force in E. coli cells. |
| Original Identifier: | oai:digitalcommons.fiu.edu:etd-1980 |
| نوع الوثيقة: | Text |
| وصف الملف: | application/pdf |
| الاتاحة: | http://digitalcommons.fiu.edu/etd/865 http://digitalcommons.fiu.edu/cgi/viewcontent.cgi?article=1980&context=etd |
| رقم الانضمام: | edsndl.fiu.edu.oai.digitalcommons.fiu.edu.etd.1980 |
| قاعدة البيانات: | Networked Digital Library of Theses & Dissertations |
| الوصف غير متاح. |