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 regulatory protein contains a single cysteine residue at position 102. DtxR readily forms inactive disulfide-linked dimers. We have used saturation site-directed mutagenesis of the cysteine codon (TGC) at position 102 in order to determine the role of this residue in metal ion binding. We show that the insertion of amino acids other than cysteine or aspartic acid into this position abolishes DtxR function both in vitro and in recombinant Escherichia coli DH5 alpha:lambda RS45toxPO/lacZ. Only those mutant alleles in which the TGC codon for Cys-102 was replaced by either TGT (Cys) or GCA (Asp) were found to direct the expression of active forms of DtxR that regulate the expression of beta-galactosidase from the toxPO/lacZ transcriptional fusion.
Evans Department of Clinical Research, Boston University Medical Center, University Hospital, MA 02118.
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D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003353:Corynebacterium diphtheriae / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005838:Genotype; D007501:Iron / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D016376:Oligonucleotides, Antisense; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; D016376:Oligonucleotides, Antisense; D011993:Recombinant Fusion Proteins; D015082:2,2'-Dipyridyl; D007501:Iron; D001616:beta-Galactosidase; D003545:Cysteine