CYSTEINE-102 IS POSITIONED IN THE METAL-BINDING ACTIVATION SITE OF THE CORYNEBACTERIUM-DIPHTHERIAE REGULATORY ELEMENT DTXR

DNA sequence analysis of dtxR has shown that the M(r) 25,316 regulator y protein contains a single cysteine residue at position 102. DtxR rea dily forms inactive disulfide-linked dimers. We have used saturation s ite-directed mutagenesis of the cysteine codon (TGC) at position 102 i n order to determine the role of this residue in metal ion binding. We show that the insertion of amino acids other than cysteine or asparti c acid into this position abolishes DtxR function both in vitro and in recombinant Escherichia coli DH5alpha:lambdaRS45toxPO/lacZ. Only thos e mutant alleles in which the TGC codon for Cys-102 was replaced by ei ther TGT (Cys) or GCA (Asp) were found to direct the expression of act ive forms of DtxR that regulate the expression of beta-galactosidase f rom the toxPO/lacZ transcriptional fusion.