Transcription-Coupled DNA Supercoiling in Escherichia Coli: Mechanisms and Biological Functions

Authors

Xiaoduo Zhi, Florida International UniversityFollow

Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Chemistry

First Advisor's Name

Fenfei Leng

First Advisor's Committee Title

Committee Chair

Second Advisor's Name

Lou W. Kim

Third Advisor's Name

Zhongwei Li

Fourth Advisor's Name

Joong-ho Moon

Fifth Advisor's Name

Kathleen Rein

Keywords

Transcription-coupled DNA supercoiling; Promoter strength; E. coli topA strains; Twin-supercoiled-domain model of transcription; Hypernegatively supercoiled DNA

Date of Defense

12-5-2012

Abstract

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.

Identifier

FI13042332

Recommended Citation

Zhi, Xiaoduo, "Transcription-Coupled DNA Supercoiling in Escherichia Coli: Mechanisms and Biological Functions" (2012). FIU Electronic Theses and Dissertations. 865.
https://digitalcommons.fiu.edu/etd/865