SanG, a transcriptional activator, controls nikkomycin biosynthesis through binding to the sanN-sanO intergenic region in Streptomyces ansochromogenes
SanG, a transcriptional activator, controls nikkomycin biosynthesis through binding to the sanN-sanO intergenic region in Streptomyces ansochromogenes
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SanG 是一种转录激活剂，通过与 Ansochromogenes 链霉菌中的 sanN-sanO 基因间区域结合来控制尼可霉素的生物合成
DOI：
10.1099/mic.0.033605-0
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发表时间：
2010-03-01
期刊：
影响因子：
2.8
通讯作者：
Tan, Huarong
中科院分区：
文献类型：
--
作者：
He, Xihong;Li, Rui;Tan, Huarong
异色链霉菌Sang是一种途径特异性调控因子，主要控制尼可霉素生物合成过程中两个转录单位的转录。SUN由三个主要功能结构域组成：N端链霉菌抗生素调节蛋白(SARP)结构域、中央ATPase结构域和C末端与LuxR家族鸟苷环化酶同源的半个结构域。Sang在大肠杆菌中以C-末端His(6)标记蛋白的形式表达。动态光散射结果表明，纯化的Sang-His(6)在溶液中为二聚体。凝胶迁移率分析表明，纯化的sAN蛋白能与含有双向Sann-Sano启动子区域的DNA片段结合。通过足迹分析确定了双向Sann-Sano启动子区域内的Sang结合位点，并确定了一个共同定向的重复序列5‘-CGGCAAG-3’。SAN在体外表现出明显的ATPase/GTPase活性，加入ATP/GTP可增强SAN与靶DNA的亲和力，但ATP/GTP的水解并不是SAN与靶DNA结合的必要条件。实时荧光定量逆转录聚合酶链式反应显示，SAN的ATPase/GTP酶结构域突变显著降低了SAN-I和SANO-V的转录水平。这些结果表明，在尼可霉素生物合成过程中，SAN的ATPase/GTPase活性调节了SAN靶基因的转录激活。
Streptomyces ansochromogenes SanG is a pathway-specific regulator that mainly controls the transcription of two transcriptional units involved in nikkomycin biosynthesis. SanG consists of three major functional domains: an N-terminal Streptomyces antibiotic regulatory protein (SARP) domain, a central ATPase domain, and a C-terminal half homologous to guanylate cyclases belonging to the LuxR family. SanG was expressed in Escherichia coli as a C-terminally His(6)-tagged protein. The purified SanG-His(6) was shown to be a dimer in solution by dynamic light scattering. An electrophoretic mobility-shift assay showed that the purified SanG protein could bind to the DNA fragment containing the bidirectional sanN-sanO promoter region. The SanG-binding sites within the bidirectional sanN-sanO promoter region were determined by footprinting analysis and identified a consensus-directed repeat sequence 5'-CGGCAAG-3'. SanG showed significant ATPase/GTPase activity in vitro, and addition of ATP/GTP enhanced the affinity of SanG for target DNA, but ATP/GTP hydrolysis was not essential for SanG binding to the target DNA. However, real-time reverse transcription PCR showed that mutation of the ATPase/GTPase domain of SanG significantly decreased the transcriptional level of sanN-I and sanO-V. These results indicated that the ATPase/GTPase activity of SanG modulated the transcriptional activation of SanG target genes during nikkomycin biosynthesis.