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Abstract：
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.
摘要：
由于耐药菌株的日益流行和HIV协同感染，结核病已重新成为对人类健康的严重威胁，迫切需要新的和更有效的治疗方法。结核分枝杆菌的PhoP-PhoR双组分系统在病原体的毒力中起重要作用，因此代表了潜在的药物靶标。研究反应调节因子PhoP对基因转录的调控机制,我们通过指数富集使用配体的系统进化鉴定了用于PhoP结合的高亲和力DNA序列。该序列包含两个由4bp间隔区隔开的7bp基序的直接重复，TCACAGC(N4)TCACAGC。通过等温滴定量热法和电泳迁移率变化测定证实了直接重复序列对PhoP结合的特异性。PhoP以高度协同的方式与直接重复序列结合为二聚体。我们发现许多先前鉴定为受PhoP调控的基因在其启动子序列中含有直接重复基序。在推定的启动子结合位点的合成DNA片段以可变的亲和力结合PhoP，这与7个bp基序中的错配数量有关，不匹配的位置，以及间隔区和侧翼序列。PhoP的磷酸化增加亲和力但不改变DNA结合的特异性。总的来说,我们的结果证实了直接重复序列是PhoP结合的共有基序，从而为鉴定不同分枝杆菌基因组中PhoP直接调节的基因铺平了道路。
