RNA polymerase sigma factor determines start-site selection but is not required for upstream promoter element activation on heteroduplex (bubble) templates
RNA polymerase sigma factor determines start-site selection but is not required for upstream promoter element activation on heteroduplex (bubble) templates
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DOI：
10.1073/pnas.94.10.4982
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发表时间：
1997-05-13
影响因子：
11.1
通讯作者：
Helmann, JD
中科院分区：
文献类型：
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作者：
Fredrick, K;Helmann, JD
细菌 RNA 聚合酶 (RNAP) 的序列选择性转录需要一个参与启动子识别和 DNA 熔解的因子，缺乏 sigma（核心酶）的 RNAP 将启动从双链体末端、切口、间隙和单链区域的 RNA 合成，我们使用包含异源双链体短区域（气泡）的 DNA 模板来比较存在和不存在各种 sigma 因子时的起始情况，使用包含 sigma(D) 依赖性鞭毛蛋白的气泡模板启动子，无论有或没有其相关的上游启动子（UP）元件，我们证明即使在不存在 sigma 的情况下，UP 元件刺激也会有效发生。这支持了一个模型，其中 UP 元件主要通过核心酶的 α 亚基起作用，以增加 RNAP 与启动子的初始关联，核心和全酶在选择用于起始的模板位置上确实有很大不同：sigma(D) 将起始限制在保守的 -10 元件下游的 8-9 个核苷酸位点，值得注意的是，sigma(A) 对起始位点选择也有显着影响，即使 sigma(A) 全酶是在相应的同源双链体上无活性，由 sigma(A) 全酶选择的起始位点位于非模板链上序列下游 8 个核苷酸，类似于 sigma(A) 识别的保守的 -10 六聚体，因此，sigma(A) 即使在单链状态下似乎也识别 -10 区域，我们建议除了其在启动子识别和起始位点解链中所描述的作用外，sigma 还定位转录起始站点。
Sequence-selective transcription by bacterial RNA polymerase (RNAP) requires a factor that participates in both promoter recognition and DNA melting, RNAP lacking sigma (core enzyme) will initiate RNA synthesis from duplex ends, nicks, gaps, and single-stranded regions, We have used DNA templates containing short regions of heteroduplex (bubbles) to compare initiation in the presence and absence of various sigma factors, Using bubble templates containing the sigma(D)-dependent flagellin promoter, with or without its associated upstream promoter (UP) element, we demonstrate that UP element stimulation occurs efficiently even in the absence of sigma. This supports a model in which the UP element acts primarily through the alpha subunit of core enzyme to increase the initial association of RNAP with the promoter, Core and holoenzyme do differ substantially in the template positions chosen for initiation: sigma(D) restricts initiation to sites 8-9 nucleotides downstream of the conserved -10 element, Remarkably, sigma(A) also has a dramatic effect on start-site selection even though the sigma(A) holoenzyme is inactive on the corresponding homoduplexes, The start sites chosen by the sigma(A) holoenzyme are located 8 nucleotides downstream of sequences on the nontemplate strand that resemble the conserved -10 hexamer recognized by sigma(A), Thus, sigma(A) appears to recognize the -10 region even in a single-stranded state, We propose that in addition to its described roles in promoter recognition and start-site melting, sigma also localizes the transcription start site.