DNA consensus sequence motif for binding response regulator PhoP, a virulence regulator of Mycobacterium tuberculosis.
DNA consensus sequence motif for binding response regulator PhoP, a virulence regulator of Mycobacterium tuberculosis.
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DOI：
10.1021/bi501019u
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发表时间：
2014-12-30
期刊：
影响因子：
2.9
通讯作者：
Wang, Shuishu
中科院分区：
文献类型：
--
作者：
He, Xiaoyuan;Wang, Shuishu
由于耐药菌株的流行以及与艾滋病毒的协同感染，结核病重新成为对人类健康的严重威胁，迫切需要新的、更有效的治疗方法。结核分枝杆菌的 PhoP-PhoR 双组分系统在病原体的毒力中发挥着重要作用，因此代表了潜在的药物靶点。为了研究响应调节因子 PhoP 调节基因转录的机制，我们通过指数富集配体的系统进化，鉴定了用于 PhoP 结合的高亲和力 DNA 序列。该序列包含由 4 bp 间隔区 TCACAGC(N4)TCACAGC 分隔的两个 7 bp 基序的同向重复。 PhoP 结合的直接重复序列的特异性通过等温滴定量热法和电泳迁移率变动测定法得到证实。 PhoP 以高度协作的方式作为二聚体与同向重复序列结合。我们发现许多之前被鉴定为受 PhoP 调控的基因在其启动子序列中含有直接重复基序。推定启动子结合位点处的合成 DNA 片段以不同的亲和力结合 PhoP，这与 7 bp 基序中的错配数量、错配位置以及间隔区和侧翼序列有关。 PhoP 的磷酸化会增加亲和力，但不会改变 DNA 结合的特异性。总的来说，我们的结果证实直接重复序列是 PhoP 结合的共有基序，从而为鉴定不同分枝杆菌基因组中 PhoP 直接调控的基因铺平了道路。
Tuberculosis has reemerged as a serious threat to human health because of the increasing prevalence of drug-resistant strains and synergetic infection with HIV, prompting an urgent need for new and more efficient treatments. The PhoP–PhoR two-component system of Mycobacterium tuberculosis plays an important role in the virulence of the pathogen and thus represents a potential drug target. To study the mechanism of gene transcription regulation by response regulator PhoP, we identified a high-affinity DNA sequence for PhoP binding using systematic evolution of ligands by exponential enrichment. The sequence contains a direct repeat of two 7 bp motifs separated by a 4 bp spacer, TCACAGC(N4)TCACAGC. The specificity of the direct-repeat sequence for PhoP binding was confirmed by isothermal titration calorimetry and electrophoretic mobility shift assays. PhoP binds to the direct repeat as a dimer in a highly cooperative manner. We found many genes previously identified to be regulated by PhoP that contain the direct-repeat motif in their promoter sequences. Synthetic DNA fragments at the putative promoter-binding sites bind PhoP with variable affinity, which is related to the number of mismatches in the 7 bp motifs, the positions of the mismatches, and the spacer and flanking sequences. Phosphorylation of PhoP increases the affinity but does not change the specificity of DNA binding. Overall, our results confirm the direct-repeat sequence as the consensus motif for PhoP binding and thus pave the way for identification of PhoP directly regulated genes in different mycobacterial genomes.
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