Different Requirements for σ Region 4 in BvgA Activation of the Bordetella pertussis Promoters Pfim3 and PfhaB
Different Requirements for σ Region 4 in BvgA Activation of the Bordetella pertussis Promoters Pfim3 and PfhaB
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DOI：
10.1016/j.jmb.2011.04.017
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发表时间：
2011-06-24
影响因子：
5.6
通讯作者：
Hinton, Deborah M.
中科院分区：
文献类型：
--
作者：
Decker, Kimberly B.;Chen, Qing;Hinton, Deborah M.
百日咳杆菌BvgA是一种全球反应调节因子，可激活毒力基因，包括编码粘附素的fim3和fhaB。在fhaB启动子P-fhaB上，BvgA结合位点位于-35启动子元件的上游，该启动子元件由RNA聚合酶(RNAP)的sigma亚基4区域识别。我们证明了Sigma区4是P-fhaB的BvgA激活所必需的，这是第二类激活的标志。相反，P-FIM3的启动子-近端BvgA结合位点包括-35区域，该区域由一串缺乏特定序列信息的胞嘧啶组成。我们证明了在P-FIM3处BvgA的激活不需要sigma区4。尽管如此，第4区突变破坏了其与核心和-35DNA的典型相互作用，影响了P-fim3的转录。使用RNAP与Sigma D581C-FeBABE切割羟基自由基将区域4定位在P-FIM3-35区域附近；使用RNAP与α276C-FeBABE或α302C-FeBABE切割也将α亚基C-末端结构域定位在-35区域内，与启动子近端的BvgA类似于P二聚体。我们的结果表明，P-FIM3的-35区含有一个类似于P二聚体的BvgA，一个Alpha亚单位C-末端结构域和Sigma区4。分子模拟表明BvgA、Sigma区4和Alpha区可能以一种新的激活机制共存于该DNA中。爱思唯尔有限公司出版。
Bordetella pertussis BvgA is a global response regulator that activates virulence genes, including adhesin-encoding fim3 and fhaB. At the fhaB promoter, P-fhaB, a BvgA binding site lies immediately upstream of the -35 promoter element recognized by Region 4 of the sigma subunit of RNA polymerase (RNAP). We demonstrate that sigma Region 4 is required for BvgA activation of P-fhaB, a hallmark of Class II activation. In contrast, the promoter-proximal BvgA binding site at P-fim3 includes the -35 region, which is composed of a tract of cytosines that lacks specific sequence information. We demonstrate that sigma Region 4 is not required for BvgA activation at P-fim3. Nonetheless, Region 4 mutations that impair its typical interactions with core and with the -35 DNA affect P-fim3 transcription. Hydroxyl radical cleavage using RNAP with sigma D581C-FeBABE positions Region 4 near the -35 region of P-fim3; cleavage using RNAP with alpha 276C-FeBABE or alpha 302C-FeBABE also positions an alpha subunit C-terminal domain within the -35 region, on a different helical face from the promoter-proximal BvgA similar to P dimer. Our results suggest that the -35 region of P-fim3 accommodates a BvgA similar to P dimer, an alpha subunit C-terminal domain, and sigma Region 4. Molecular modeling suggests how BvgA, sigma Region 4, and alpha might coexist within this DNA in a conformation that suggests a novel mechanism of activation. Published by Elsevier Ltd.