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

The F Factor TraY protein is a sequence-specific DNA-binding protein requir ed for efficient conjugal transfer. Genetic and biochemical studies indicat e 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 n icking of F Factor in preparation for transfer. TraY is thought to belong t o the ribbon-helix-helix family of transcription factors. These proteins co ntact DNA using residues of an antiparallel beta-sheet. We engineered and c haracterized six TraY mutants each having a single potential beta-sheet DNA contact residue replaced with Ala. Most TraY mutants had significantly red uced affinity for the TraY oriT binding site while possessing near wild-typ e stability and nonspecific DNA recognition. These results indicate that Tr aY beta-sheet residues participate in DNA recognition, and support inclusio n of TraY in the ribbon-helix-helix family.