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

Abstract pSM19035‐encoded dimeric regulator omega (ω 2 ) belongs to the MetJ/Arc family of ribbon–helix–helix DNA binding proteins. ω 2 binds to a set of unspaced 7‐base pair repeat units with sequence 5′‐ A / T ATCAC A / T ‐3′. Protein ω 2 and its variant ω 2 ΔN18, 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 ω 2 and ω 2 ΔN18 bind to operator DNA in the major groove and contact thymine, adenine and guanine residues. The mode of ω 2 or ω 2 ΔN18 interaction with operator DNA is not affected by the orientation and number of heptads. The Raman data of ω 2 T29A, an ω 2 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 ω 2 protein to its cognate site. The Raman data strongly support the notion that the 18 N ‐terminal amino acids are not required for ω 2 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 ω 2 to one of the bases in the central 5′‐TCA‐3′/5′‐TGA‐3′ stretch of the 7‐bp binding site.