Specific DNA recognition by F factor TraY involves β-sheet residues

Abstract

The F Factor TraY protein is a sequence-specific DNA-binding protein required for efficient conjugal transfer. Genetic and biochemical studies indicate that TraY has two functional roles in conjugation. TraY binds to the P(Y) promoter to up-regulate transcription of tra genes. TraY also binds to the plasmid origin of transfer (oriT), serving as an accessory protein in the nicking of F Factor in preparation for transfer. TraY is thought to belong to the ribbon-helix-helix family of transcription factors. These proteins contact DNA using residues of an antiparallel β-sheet. We engineered and characterized six TraY mutants each having a single potential β-sheet DNA contact residue replaced with Ala. Most TraY mutants had significantly reduced affinity for the TraY oriT binding site while possessing near wild- type stability and nonspecific DNA recognition. These results indicate that TraY β-sheet residues participate in DNA recognition, and support inclusion of TraY in the ribbon-helix-helix family.