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Supercoiling and integration host factor change the DNA conformation and alter the flow of convergent transcription
N P Higgins1, D A Collier, M W Kilpatrick
1Department of Biochemistry, School of Medicine, University of Alabama, Birmingham 35294.
Abstract:
Transcription of bacteriophage Mu is modulated by its repressor, by negative supercoiling, and by the Escherichia coli protein integration host factor (IHF). Two converging Mu promoters regulate lytic and lysogenic development. The influence of IHF on these convergent promoters depended on the DNA conformation. When Mu operator DNA changed from the relaxed to the negative supercoiled form, IHF changed from a stimulatory factor to an inhibitor of the repressor promoter, and the ratio of the lytic transcript relative to the repressor transcript increased by 40-fold. Flexibility in Mu operator DNA was demonstrated by an unusual supercoil-induced DNA conformation, which was detectable by chemical modification with bromoacetaldehyde or digestion with P1 nuclease. IHF binding adjacent to this site induced dramatic bending of Mu DNA. A topological model we call a superloop is proposed to explain the effect of IHF on Mu transcription in vitro and on the lytic-lysogeny decision of the virus grown in IHF and gyrase mutants.
Insights
Bacteriophage Mu transcription relies on DNA supercoiling and Integration Host Factor (IHF). DNA conformation changes alter IHF
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Bacteriophage Mu transcription is regulated by its repressor, DNA supercoiling, and the E. coli protein Integration Host Factor (IHF).
- Two converging promoters control the lytic and lysogenic development of bacteriophage Mu.
Purpose of the Study:
- To investigate the role of DNA conformation in modulating the influence of IHF on bacteriophage Mu transcription.
- To elucidate the mechanism by which IHF affects Mu's lytic-lysogenic decision.
Main Methods:
- Analysis of bacteriophage Mu transcription in vitro under varying DNA supercoiling conditions.
- Chemical modification (bromoacetaldehyde) and enzymatic digestion (P1 nuclease) to detect DNA conformational changes.
- Assessment of IHF binding and its effect on Mu DNA bending.
Main Results:
- IHF's role shifted from a stimulatory factor to an inhibitor of the repressor promoter upon transition from relaxed to negatively supercoiled DNA.
- A 40-fold increase in the ratio of lytic to repressor transcripts was observed with negative supercoiling.
- Supercoil-induced DNA conformational changes and IHF-mediated DNA bending were demonstrated.
Conclusions:
- DNA conformation significantly impacts IHF's regulatory function on bacteriophage Mu transcription.
- A 'superloop' topological model explains IHF's effect on Mu transcription and the lytic-lysogeny decision.
- The findings provide insights into viral development regulation influenced by host factors and DNA topology.