DETERMINATION OF THE MINIMAL ESSENTIAL NUCLEOTIDE-SEQUENCE FOR DIPHTHERIA TOX REPRESSOR BINDING BY IN-VITRO AFFINITY SELECTION

The expression of diphtheria toxin in lysogenic toxigenic strains of C orynebacterium diphtheriae is controlled by the heavy metal ion-activa ted regulatory protein DtxR. In the presence of divalent heavy metal i ons, DtxR specifically binds to the diphtheria ton operator and protec ts a 27-bp interrupted palindromic sequence from DNase I digestion. To determine the consensus DNA sequence for DtxR binding, we have used g el electrophoresis mobility-shift assay and polymerase chain reaction (PCR) amplification for in vitro affinity selection of DNA binding seq uences 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 am plification, we isolated and characterized a family of DNA sequences t hat 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 ton operator. The DN A sequence analysis of 21 unique in vitro affinity-selected binding si tes has revealed the minimal essential nucleotide sequence for DtxR bi nding to be a 9-bp palindrome separated by a single base pair.