Isolation and characterization of iron-independent positive dominant mutants of the diphtheria toxin repressor DtxR.
Isolation and characterization of iron-independent positive dominant mutants of the diphtheria toxin repressor DtxR.
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DOI：
10.1073/pnas.95.25.14985
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发表时间：
1998-12
影响因子：
11.1
通讯作者：
L. Sun;J. vanderSpek;J. Murphy
中科院分区：
文献类型：
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作者：
L. Sun;J. vanderSpek;J. Murphy
众所周知，白喉毒素阻遏物DtxR的功能活性由铁控制，铁作为通过该调节元件激活靶DNA结合所必需的必需辅因子。在这篇文章中，我们描述了一系列独特的DtxR突变体的分离和表征，这些突变体是组成型活性的，并且抑制白喉毒素启动子/操作子-lacZ转录融合的β-半乳糖苷酶的表达，即使在没有铁的情况下。这些自激活突变体的DtxR（SAD）的分离，通过使用一个积极的选择系统的功能dtxR等位基因和目标DNA操纵位点的克隆。在四个独立分离的SAD突变体中，发现两个（SAD 2和SAD 11）在其各自的C-末端SH 3样结构域中携带单个错义突变（E175 K）。与此相反，突变等位基因编码SAD 3被发现携带总共六个错义突变分布在整个N-和C-末端结构域的阻遏物。部分二倍体分析的菌株携带天然dtxR和等位基因编码SAD 2或SAD 3表明，这些铁非依赖性突变体具有积极的显性表型，在调节β-半乳糖苷酶的表达从白喉毒素启动子/操作子-lacZ转录融合。
It is well known that the functional activity of the diphtheria toxin repressor DtxR is controlled by iron, which serves as an essential cofactor necessary for activation of target DNA binding by this regulatory element. In this communication, we describe the isolation and characterization of a unique series of DtxR mutants that are constitutively active and repress the expression of beta-galactosidase from a diphtheria tox promoter/operator-lacZ transcriptional fusion, even in the absence of iron. These self-activating mutants of DtxR (SAD) were isolated through the use of a positive selection system for the cloning of functional dtxR alleles and target DNA operator sites. Of the four independently isolated SAD mutants that were characterized, two (SAD2 and SAD11) were found to carry a single missense mutation (E175K) in their respective C-terminal SH3-like domains. In contrast, the mutant allele encoding SAD3 was found to carry a total of six missense mutations distributed throughout the N- and C-terminal domains of the repressor. Partial diploid analysis of strains carrying both native dtxR and alleles encoding either SAD2 or SAD3 demonstrate that these iron-independent mutants possess a positive dominant phenotype in the regulation of beta-galactosidase expression from a diphtheria tox promoter/operator-lacZ transcriptional fusion.