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Coupling DNA supercoiling to transcription in defined protein systems
Fenfei Leng1, Luciana Amado, Roger McMacken
1Department of Chemistry & Biochemistry, Florida International University, Miami, Florida 33199, USA. lengf@fiu.edu
The Journal of Biological Chemistry
|September 3, 2004
Summary
Transcription-coupled DNA supercoiling (TCDS) in vitro occurs via two pathways. A potent twin-domain mechanism, dominant with long transcripts and bending proteins, efficiently generates high superhelicity levels.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA supercoiling is crucial for DNA metabolism.
- Transcription-coupled DNA supercoiling (TCDS) is known to occur in vivo.
- Establishing an in vitro system for TCDS via the twin-domain mechanism has been challenging.
Purpose of the Study:
- To investigate the mechanistic pathways of TCDS in vitro.
- To characterize the conditions favoring the twin-domain mechanism of TCDS.
- To compare in vitro TCDS with in vivo mechanisms.
Main Methods:
- Utilized defined protein systems with T7 RNA polymerase and DNA gyrase.
- Employed specifically designed plasmid DNA templates to control RNA transcripts.
- Assessed TCDS activity under varying conditions, including presence of RNase H and DNA-bending proteins.
Main Results:
- Identified two distinct in vitro pathways for TCDS.
- One pathway, R-loop dependent, is suppressed by RNase H or HU protein.
- A second, potent pathway, dominant with long transcripts (>3 kb) and bending proteins, is RNase H resistant and resembles the twin-domain mechanism.
Conclusions:
- TCDS in vitro proceeds through at least two independent pathways.
- The twin-domain mechanism can be reconstituted in vitro under specific conditions.
- This reconstituted system efficiently generates high levels of DNA superhelicity, relevant to in vivo processes.