Purification, characterization, and gene expression of all sigma factors of RNA polymerase in a cyanobacterium
Purification, characterization, and gene expression of all sigma factors of RNA polymerase in a cyanobacterium
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DOI：
10.1016/s0022-2836(02)01242-1
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发表时间：
2003-01-31
影响因子：
5.6
通讯作者：
Shirai, M
中科院分区：
文献类型：
--
作者：
Imamura, S;Yoshihara, S;Shirai, M
RNA聚合酶（RNAP）σ因子基因和蛋白质的表达被表征为了解它们在单细胞蓝藻集胞藻中的功能的第一步。 PCC 6803，可以进行光合作用。从大肠杆菌细胞中过量产生并纯化所有九个 sigma 因子（第 1 组，SigA；第 2 组，SigB 至 SigE；第 3 组，SigF 至 SigI）和每个 RNAP 核心亚基（RpoA、RpoB、RpoC1 和 RpoC2），然后制备多克隆抗体。 Western blot 和引物延伸分析显示，在稳态生长条件下，第 1 组和第 2 组 sigma 因子的细胞内水平范围为每微克总蛋白 0.9 fmol 至 9.3 fmol，并且观察到生长阶段依赖性或组成型转录本。有趣的是，在正常生理条件下没有检测到第 3 组西格玛因子蛋白，而它们的转录物却很强大，这意味着翻译衰减和/或蛋白质不稳定的可能调节。系统发育分析还表明，蓝藻的第3组西格玛因子同源物是保守的，但它们之间存在进化或功能分歧。体外和体内结果表明，高光响应性 SigD 表达及其启动子对光合作用基因 psbA 的识别具有重要证据。另一方面，观察到自动调节的 sigB 转录、细胞热休克后 SigB 表达显着增加，以及 SigB 的特异性启动子识别以及热休克 hspA 启动子上其他 sigma 因子的冗余。这些发现清楚地表明 SigB 是一种热休克反应性西格玛因子。本文还讨论了相关西格玛因子基因的独特启动子结构和表达。 (C) 2003 Elsevier Science Ltd. 保留所有权利。
The expression of RNA polymerase (RNAP) sigma factor genes and proteins was characterized as a first step toward understanding their functions in a unicellular cyanobacterium Synechocystis sp. PCC 6803, which can perform photosynthesis. All nine sigma factors (group 1, SigA; group 2, SigB to SigE; and group 3, SigF to SigI) and each RNAP core subunit (RpoA, RpoB, RpoC1 and RpoC2) were overproduced and purified from Escherichia coli cells, then polyclonal antibodies were prepared. Western blot and primer extension analyses revealed that the intracellular levels of group 1 and 2 sigma factors ranged from 0.9 fmol to 9.3 fmol per micro-gram of the total protein under conditions of steady-state growth, and that growth phase-dependent or constitutive transcripts were observed. Interestingly, no group 3 sigma factor proteins were detected under normal physiological conditions whereas their transcripts were robust, implying a possible regulation of translational attenuation and/or protein instability. Phylogenetic analysis also revealed that group 3 sigma factor homologues of cyanobacteria are conserved with evolutionary or functionary divergence among them. In vitro and in vivo results indicated significant evidence of high-light responsive SigD expression and its promoter recognition of the photosynthesis gene, psbA. On the other hand, autoregulated sigB transcription, a dramatically increased SigB expression upon the exposure of cells to heat-shock, and specific promoter recognition by SigB with redundancy of other sigma factors on the heat-shock hspA promoter were observed. These findings clearly indicated that SigB is a heat-shock responsive sigma factor. The unique promoter architecture and expression of the relevant sigma factor gene are also discussed herein. (C) 2003 Elsevier Science Ltd. All rights reserved.