Binding of regulatory protein omega from Streptococcus pyogenes plasmid pSM19035 to direct and inverted 7‐base pair repeats of operator DNA

pSM19035‐encoded dimeric regulator omega (ω₂) belongs to the MetJ/Arc family of ribbon–helix–helix DNA binding proteins. ω₂ binds to a set of unspaced 7‐base pair repeat units with sequence 5′‐^A/_TATCAC^A/_T‐3′. Protein ω₂ and its variant ω₂ΔN18, lacking the first 18 N‐terminal amino acids, bind poorly to one heptad DNA, but the affinity to DNA markedly increases with two and more heptads organized in direct or inverted orientation. Raman difference spectra indicate that ω₂ and ω₂ΔN18 bind to operator DNA in the major groove and contact thymine, adenine and guanine residues. The mode of ω₂ or ω₂ΔN18 interaction with operator DNA is not affected by the orientation and number of heptads. The Raman data of ω₂T29A, an ω₂ variant with Ala instead of Thr at position 29 of the β‐sheet, show a sequence‐independent binding mode to DNA, which differs from the binding mode of wt ω₂ protein to its cognate site. The Raman data strongly support the notion that the 18 N‐terminal amino acids are not required for ω₂ activity, whereas Thr29 plays an essential role for operator DNA binding. The data suggest the formation of a hydrogen bond between Thr29 of wild‐type ω₂ to one of the bases in the central 5′‐TCA‐3′/5′‐TGA‐3′ stretch of the 7‐bp binding site. Copyright © 2006 John Wiley & Sons, Ltd.