Binding of PhoP to promoters of phosphate-regulated genes in Streptomyces coelicolor:: identification of PHO boxes
Binding of PhoP to promoters of phosphate-regulated genes in Streptomyces coelicolor:: identification of PHO boxes
复制标题
DOI：
10.1111/j.1365-2958.2005.04631.x
复制
发表时间：
2005-06-01
影响因子：
3.6
通讯作者：
Martín, JF
中科院分区：
文献类型：
--
作者：
Sola-Landa, A;Rodríguez-García, A;Martín, JF
天蓝色链霉菌中磷酸盐调控基因的调控是由双组分系统PhoR-PhoP介导的。当与报告基因xylE偶联时，pstS、phoRP和phoU启动子显示出对磷酸调节非常敏感。通过引物延伸鉴定pstS、phoRP和phoU启动子的转录起始点。phoRP显示了无领导的转录本。反应调节（DNA结合）PhoP蛋白在大肠杆菌中过表达并纯化为GST-PhoP融合蛋白。PhoP的DNA结合结构域（DBD）也以类似的方式获得。发现PhoP及其截短的DBD结构域都以高亲和力结合到pstS和phoRP-phoU启动子的上游区域，靠近这些启动子各自的-35序列。DNA酶I保护研究揭示了在pstS启动子的有义链中的29 bp受保护的延伸，其包括两个11 bp的直接重复单元。双向phoRP-phoU启动子区域的足迹显示了一个51 bp的保护序列，包括四个直接重复单元，其中两个与pstS启动子中的保护序列具有高度相似性。PHO盒已经通过在这些启动子区域中发现的六个直接重复单元的比对来鉴定。每个同向重复单元调整为共有序列G（G/T）TCAYYYR（G/C）G。
The control of phosphate-regulated genes in Streptomyces coelicolor is mediated by the two-component system PhoR-PhoP. When coupled to the reporter xylE gene the pstS, phoRP and phoU promoters were shown to be very sensitive to phosphate regulation. The transcription start points of the pstS, the phoRP and the phoU promoters were identified by primer extension. phoRP showed a leaderless transcript. The response-regulator (DNA-binding) PhoP protein was overexpressed and purified in Escherichia coli as a GST-PhoP fused protein. The DNA-binding domain (DBD) of PhoP was also obtained in a similar manner. Both PhoP and its truncated DBD domain were found to bind with high affinity to an upstream region of the pstS and phoRP-phoU promoters close to the -35 sequence of each of these promoters. DNase I protection studies revealed a 29 bp protected stretch in the sense strand of the pstS promoter that includes two 11 bp direct repeat units. Footprinting of the bidirectional phoRP-phoU promoter region showed a 51 bp protected sequence that encompasses four direct repeat units, two of them with high similarity to the protected sequences in the pstS promoter. PHO boxes have been identified by alignment of the six direct repeat units found in those promoter regions. Each direct repeat unit adjusts to the consensus G(G/T)TCAYYYR(G/C)G.