title: Determination of the minimal essential nucleotide sequence for diphtheria tox repressor binding by in vitro affinity selection.
abstract: The expression of diphtheria toxin in lysogenic toxigenic strains of Corynebacterium diphtheriae is controlled by the heavy metal ion-activated regulatory protein DtxR. In the presence of divalent heavy metal ions, DtxR specifically binds to the diphtheria tox operator and protects a 27-bp interrupted palindromic sequence from DNase I digestion. To determine the consensus DNA sequence for DtxR binding, we have used gel electrophoresis mobility-shift assay and polymerase chain reaction (PCR) amplification for in vitro affinity selection of DNA binding sequences from a universe of 6.9 x 10(10) variants. After 10 rounds of in vitro affinity selection, each round coupled with 30 cycles of PCR amplification, we isolated and characterized a family of DNA sequences that function as DtxR-responsive genetic elements both in vitro and in vivo. Moreover, these DNA sequences were found to bind activated DtxR with an affinity similar to that of the wild-type tox operator. The DNA sequence analysis of 21 unique in vitro affinity-selected binding sites has revealed the minimal essential nucleotide sequence for DtxR binding to be a 9-bp palindrome separated by a single base pair.
authors.affiliation: Department of Biochemistry, Boston University School of Medicine, MA 02118.
authors.forename: X
authors.initials: X
authors.lastname: Tao
authors.forename: J R
authors.initials: JR
authors.lastname: Murphy
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D016384:Consensus Sequence; D003353:Corynebacterium diphtheriae / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C075586:DtxR protein, Corynebacterium diphtheriae; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012097:Repressor Proteins