DNA Recognition and Wrapping by Escherichia coli RcnR

Escherichia coli RcnR is a founding member of a recently discovered large and widespread structural family of bacterial transcription factors that are predicted to respond to a variety of environmental stresses. RcnR directly regulates transcription of the gene encoding the RcnA nickel and cobalt efflux protein by coordination of DNA-binding and metal-binding activities. A crystal structure of a Cu(I)-sensing homolog from Mycobacterium tuberculosis did not reveal how the novel all-α-helical fold of this protein family interacts with DNA because it lacks a well-characterized DNA-binding motif. In this study, we investigated the biophysical properties of the RcnR–DNA interaction using isothermal titration calorimetry and footprinting techniques. We found that an RcnR tetramer recognizes a TACT-G6-N-AGTA motif, of which there are two in the rcnA–rcnR intergenic region. G-tracts are found in many predicted binding sites of other RcnR/CsoR proteins, and here we show that they endow A-form DNA characteristics to the RcnR operator sites. Interestingly, RcnR also interacts nonspecifically with the ∼ 50 base pairs flanking the core binding site, resulting in DNA wrapping and the introduction of a single negative supercoil into plasmid DNA. Comparisons with other RcnR/CsoR proteins reveal likely key differences in DNA binding among members of this family that result from variations in the number and sequence of operator sites.