Determination of the minimal essential nucleotide sequence for diphtheria tox repressor binding by in vitro affinity selection.
Determination of the minimal essential nucleotide sequence for diphtheria tox repressor binding by in vitro affinity selection.
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DOI：
10.1073/pnas.91.20.9646
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发表时间：
1994-09
影响因子：
11.1
通讯作者：
X. Tao;J. Murphy
中科院分区：
文献类型：
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作者：
X. Tao;J. Murphy
白喉棒状杆菌溶原性产毒菌株中白喉毒素的表达受重金属离子激活调节蛋白DtxR的调控。在二价重金属离子存在的情况下，DtxR特异性地与白喉毒素操纵子结合，并保护一个27bp的中断回文序列免受DNase I的消化。为了确定DtxR结合的共同DNA序列，我们使用凝胶电泳迁移率改变分析和聚合酶链式反应(PCR)扩增从6.9×10(10)个变异体中进行DNA结合序列的体外亲和选择。经过10轮体外亲和筛选，每轮再加上30个循环的PCR扩增，我们分离并鉴定了一系列在体外和体内都具有DtxR反应遗传元件的DNA序列。此外，这些DNA序列被发现以与野生型TOX操纵子相似的亲和力结合激活的DtxR。21个独特的体外亲和选择结合位点的DNA序列分析表明，DtxR结合的最小必需核苷酸序列是由单个碱基对分隔的9个碱基对的回文序列。
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.