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 PY 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 beta-sheet. We engineered and characterized six TraY mutants each having a single potential beta-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 beta-sheet residues participate in DNA recognition, and support inclusion of TraY in the ribbon-helix-helix family.
Highlights
Based on a shared pattern of mainly hydrophobic amino acids (Fig. 1A), Bowie and Sauer [8] assigned TraY to the ribbon-helix-helix family of transcription factors
We engineered a series of TraY mutants with Ala substitutions within the TraY -sheet region, and characterized the DNA recognition and protein stability of these mutants
Sites of amino acid substitutions were selected based upon the sequence alignment of TraY with other members of the ribbonhelix-helix family (Fig. 1A)
Summary
Bacterial Strains, and Plasmids—Restriction endonucleases were obtained from New England Biolabs and Stratagene. Plasmid purified from transformants was screened by restriction enzyme digestion Both strands of mutant traY genes were fully sequenced prior to protein expression and functional analysis. Varying concentrations of TraY or TraY mutant were incubated for 90 min with 22 pM (final concentration) 32P-end-labeled specific oriT binding site oligonucleotide. Data were converted to fraction of labeled DNA bound and plotted versus protein concentration. Multiple experiments were fit simultaneously with the equation: ϭ 1/(1 ϩ KD/[protein]), where is fraction DNA bound, using Kaleidagraph 3.0 (Synergy Software) Affinities for both specific and nonspecific DNA binding sites were measured using a competition assay. The concentration of the radiolabeled specific oriT binding site used for TraY and most mutants was equal to the measured KD of that protein. Data from multiple experiments were normalized and combined into a single fit
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