Molecular basis of transcription initiation in Archaea
Molecular basis of transcription initiation in Archaea
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DOI：
10.4161/trns.1.2.13189
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发表时间：
2010-01-01
影响因子：
3.6
通讯作者：
Hoenger, Andreas
中科院分区：
文献类型：
--
作者：
De Carlo, Sacha;Lin, Shih-Chieh;Hoenger, Andreas
与真核生物相比，古细菌的转录起始机制（通常称为预起始复合体）相对简单。古细菌 PIC 由 TFIIB 直系同源物 TFB、TBP 和 11 亚基 RNA 聚合酶 (RNAP) 组成。整个古菌 PIC 的尺寸相对较小，因此适合进行结构分析。使用从嗜热火球菌中纯化的 RNAP、TFB 和 TBP，我们在 65 摄氏度下组装了具有生化活性的 PIC。通过实施交联技术和尺寸排阻色谱分离了完整的古菌 PIC，并使用电子显微镜和单颗粒重建技术确定了该 440 kDa 组装体的结构。将差异图谱与各个子域的晶体结构对接相结合，TBP和TFB定位在大分子PIC内。 TBP/TFB 在大 RpoB 亚基和 RNAP 中央裂口后面的 RpoD/L“脚”结构域附近组装。该位置模拟了酵母 TBP 和 TFIIB 与酵母 RNAP II 的复合物。总的来说，这些结果定义了古细菌转录机制的结构组织，并提出了一个保守的核心 PIC 架构。
Compared with eukaryotes, the archaeal transcription initiation machinery-commonly known as the Pre-Initiation Complex-is relatively simple. The archaeal PIC consists of the TFIIB ortholog TFB, TBP and an 11-subunit RNA polymerase (RNAP). The relatively small size of the entire archaeal PIC makes it amenable to structural analysis. Using purified RNAP, TFB and TBP from the thermophile Pyrococcus furiosus, we assembled the biochemically active PIC at 65 degrees C. The intact archaeal PIC was isolated by implementing a cross-linking technique followed by size-exclusion chromatography, and the structure of this 440 kDa assembly was determined using electron microscopy and single-particle reconstruction techniques. Combining difference maps with crystal structure docking of various sub-domains, TBP and TFB were localized within the macromolecular PIC. TBP/TFB assemble near the large RpoB subunit and the RpoD/L "foot" domain behind the RNAP central cleft. This location mimics that of yeast TBP and TFIIB in complex with yeast RNAP II. Collectively, these results define the structural organization of the archaeal transcription machinery and suggest a conserved core PIC architecture.