Abstract
The TraY protein is required for efficient bacterial conjugation by Escherichia coli F factor. TraY has two functional roles: participating in the "relaxosome," a protein-DNA complex that nicks one strand of the F factor plasmid, and up-regulating transcription from the traYI promoter. The traY gene was cloned, and the TraY protein was expressed, purified, and characterized. TraY has a mixed alpha-helix and beta-sheet secondary structure as judged by its circular dichroism spectrum, is monomeric, and undergoes reversible urea denaturation with delta Gu = 6 kcal/mol at 25 degrees C. The kinetics of protein unfolding and refolding, as measured by changes in fluorescence, are complex, suggesting the presence of intermediates or of heterogeneity in the folding reaction. TraY has been classified as a member of the ribbon-helix-helix family of transcription factors but is unusual in appearing to have tandem repeats of the beta alpha alpha motif in the same polypeptide chain. The data presented here show that folding and assembly of the functional (beta alpha alpha)2 unit occurs as an intramolecular reaction and not by cross-folding between different polypeptide chains.
Highlights
In a sequence-specific manner and are known to bend or distort the DNA (10 –12)
We describe the biophysical characterization of TraY as a first step in determining the structural basis for the dual activities of TraY
TraY Purification—The gene encoding F factor TraY was amplified by PCR, cloned into an expression vector, expressed in E. coli strain BL21(DE3), and purified
Summary
Cloning of traY Gene—The traY gene was amplified by PCR1 using genomic DNA from E. coli Hfr strain CAG5051 [16]. Equilibrium Unfolding and Refolding—For urea denaturation experiments, equimolar solutions of TraY were made using 20 mM sodium phosphate (pH 7.4), 100 mM NaCl, 1 mM EDTA, 5 mM -mercaptoethanol, and either 0 or 9.5 M urea. A solution of TraY denatured in buffer plus 8 M urea was mixed with solutions containing 0 –2.4 M urea in a 1:5 ratio to give final urea concentrations of 1.3–3.3 M. A solution of TraY in 4 M urea was mixed in a 1:5 ratio with solutions containing 7.6 –10 M urea to give final urea concentrations of 7–9 M. For jumps into the refolding/unfolding transition zone, solutions of 6 M TraY in buffer plus 4 M urea (native) or buffer plus 8 M urea (denatured) were mixed with solutions of higher or lower urea to give final concentrations of 5– 6 M. A partial specific volume of 0.7407 was calculated according to Cohn and Edsall [23] for use in the analysis
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