Two roles for integration host factor at an enhancer-dependent nifA promoter

Area of Science:

• Microbiology
• Molecular Biology
• Gene Regulation

Background:

• Prokaryotic transcription control often involves regulatory proteins interacting directly with RNA polymerase.
• For sigma54 RNA polymerase, distal enhancers engage the polymerase through DNA looping.
• The Herbaspirillum seropedicae nifA promoter is sigma54-dependent and activated during nitrogen limitation.

Purpose of the Study:

• To investigate the roles of potential enhancers and binding sites for NTRC, NIFA, IHF, and sigma54-holoenzyme at the Hs nifA promoter.
• To explore the involvement of these sites in promoter regulation.
• To elucidate the differential effects of IHF on NTRC- and NIFA-mediated promoter activation.

Main Methods:

• Identification of potential enhancer and binding sites for regulatory proteins and sigma54-holoenzyme.
• DNA footprinting assays to confirm protein binding to identified sites.
• In vitro transcription assays to assess promoter activation by NTRC and NIFA under varying IHF concentrations.

Main Results:

• DNA footprinting confirmed functional protein binding sites for NTRC, NIFA, IHF, and sigma54-holoenzyme.
• IHF significantly stimulates in vitro activation of the Hs nifA promoter by NTRC.
• Conversely, IHF strongly inhibits NIFA-mediated activation and high NIFA levels inhibit NTRC activation in an IHF-dependent manner.

Conclusions:

• IHF plays a dual role at the Hs nifA promoter, acting both positively and negatively.
• IHF ensures that transcription activation is restricted to the NTRC activator and a specific signal transduction pathway.
• This regulation prevents potentially destabilizing autoactivation by NIFA and fine-tunes nifA promoter activity.