The CTXphi repressor RstR binds DNA cooperatively to form tetrameric repressor-operator complexes

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• CTX phage integrates into Vibrio cholerae, forming stable lysogens.
• The phage repressor RstR controls gene expression from the rstA promoter.

Purpose of the Study:

• To investigate the DNA-binding mechanism and oligomerization state of the CTX phage repressor, RstR.
• To elucidate the molecular basis of RstR-mediated gene repression.

Main Methods:

• Purification of His-tagged RstR.
• DNA binding assays using DNase I footprinting.
• Oligomerization studies via gel permeation chromatography and cross-linking.
• Electrophoretic mobility shift assays (EMSAs) to determine RstR-DNA complex formation.

Main Results:

• RstR binds to three operator sites within the rstA promoter region.
• DNase I footprints suggest RstR binds DNA as a dimer-of-dimers (tetramer).
• RstR forms dimers and tetramers in solution and is tetrameric when bound to DNA.
• RstR binding to the O1 operator site follows a cooperative model, involving tetrameric complex formation.

Conclusions:

• RstR functions as a tetramer to repress CTX phage gene expression.
• Protein-protein contacts between RstR dimers are crucial for high-affinity operator binding.
• This tetrameric binding mechanism provides insights into phage-host genetic regulation.