Specific binding of SsoII DNA methyltransferase to its promoter region provides the regulation of SsoII restriction-modification gene expression

Abstract

The regulation of the SsoIIl restriction-modification system from Shigella sonnei was studied in vivo and in vitro. In lacZ fusion experiments, SsoII methyltransferase (M.SsoII) was found to repress its own synthesis but stimulate expression of the cognate restriction endonuclease (ENase). The N-terminal 72 amino acids of M.SsoII, predicted to form a helix-turn-helix (HTH) motif, was found to be responsible for the specific DNA-binding and regulatory function of M.SsoII. Similar HTH motifs are predicted in the N-terminus of a number of 5-methylcytosine methyltransferases, particularly M.EcoRII, M.dcm and M.MspI, of which the ability to regulate autogenously has been proposed. In vitro, the binding of M.SsoII to its target DNA was investigated using a mobility shift assay. M.SsoII forms a specific and stable complex with a 140 bp DNA fragment containing the promoter region of SsoII R-M system. The dissociation constant (K(d)) was determined to be 1.5 x 10-8 M. DNasel footprinting experiments demonstrated that M.SsoII protects a 48-52 bp region immediately upstream of the M.SsoII coding sequence which includes the predicted -10 promoter sequence of M.SsoII and the -10 and -35 sequences of R.SsoII.