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Analysis of diphtheria toxin repressor‐operator interactions and characterization of a mutant repressor with decreased binding activity for divalent metals
Author(s) -
Schmitt Michael P.,
Holmes Randall K.
Publication year - 1993
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1993.tb01679.x
Subject(s) - repressor , corynebacterium diphtheriae , diphtheria toxin , biology , mutant , operon , dna , dna footprinting , binding site , biochemistry , microbiology and biotechnology , dna binding protein , gene , toxin , diphtheria , gene expression , transcription factor , vaccination , immunology
Summary The diphtheria toxin repressor (DtxR) is an Fe 2+ ‐activated protein with sequence‐specific DNA‐binding activity for the diphtheria toxin (tox) operator. Under high‐iron conditions in Corynebacterium diphtheriae , DtxR represses toxin and siderophore biosynthesis as well as iron uptake. DtxR and a mutant repressor with His–47 substituted for Arg–47, designated DtxR‐R47H, were purified and compared. Six different divalent cations (Cd 2+ , Co 2+ , Fe 2+ , Mn 2+ , Ni 2+ , and Zn 2+ ) activated the sequence‐specific DNA‐binding activity of DtxR and enabled it to protect the fox operator from DNase I digestion, but Cu 2+ failed to activate DtxR. Hydroxyl radical footprinting experiments indicated that DtxR binds symmetrically about the dyad axis of the tox operator. Methylation protection experiments demonstrated that DtxR binding alters the susceptibility to methylation of three G residues within the AT‐rich tox operator. These findings suggest that two or more monomers of DtxR are involved in binding to the tox operator, with symmetrical DNA‐protein interactions occurring at each end of the palindromic operator. In this regard, DtxR resembles several other well‐characterized prokaryotic repressor proteins but differs dramatically from the Fe 2+ ‐activated ferric uptake repressor protein (Fur) of Escherichia coli. The concentration of Co 2+ required to activate DtxR‐R47H was at least 10‐foid greater than that needed to activate DtxR, but the sequence‐specific DNA binding of activated DtxR‐R47H was indistinguishable from that of wild‐type DtxR. The markedly deficient repressor activity of DtxR‐R47H is consistent with a significant decrease in its binding activity for divalent cations.