Science2013-11-10 2:19 AM

Architecture of an RNA Polymerase II Transcription Pre-Initiation Complex RNAⅡ型聚合酶转录前起始复合物的结构

Abstract The protein density and arrangement of subunits of a complete, 32-protein, RNA polymerase II (pol II) transcription pre-initiation complex (PIC) were determined by means of cryogenic electron microscopy and a combination of chemical cross-linking and mass spectrometry. The PIC showed a marked division in two parts, one containing all the general transcription factors (GTFs) and the other pol II. Promoter DNA was associated only with the GTFs, suspended above the pol II cleft and not in contact with pol II. This structural principle of the PIC underlies its conversion to a transcriptionally active state; the PIC is poised for the formation of a transcription bubble and descent of the DNA into the pol II cleft. 论文摘要 一个完整的有32个蛋白的RNAⅡ型聚合酶转录前起始复合物(PIC)亚基的蛋白密度和排列分布是借助于低温电子显微镜以及化学交联和质谱分析相结合的技术手段而得以确定的。该前起始复合物表现出两部分的显著划分,一部分包含着所有通用转录因子(GTFs),另一部分包含着Ⅱ型聚合酶。启动子DNA只与通用转录因子发生关联,在Ⅱ型聚合酶裂口前,它就停止前进不和Ⅱ型聚合酶发生任何接触。前起始复合物的这一结构性原理为它转变成转录活性状态提供了基础;前起始复合物为转录空泡的成形和DNA降落进入Ⅱ型聚合酶裂口中作了准备。 Introduction RNA polymerase II (pol II) is capable of RNA synthesis but is unable to recognize a promoter or to initiate transcription. For these essential functions, a set of general transcription factors (GTFs)—termed TFIIB, -D, -E, -F, and -H—is required. The GTFs escort promoter DNA through the stages of recruitment to pol II, unwinding to create a transcription bubble, descent into the pol II cleft, and RNA synthesis to a length of 25 residues and transition to a stable elongating complex. The structural basis for these transactions is largely unknown. Only TFIIB has been solved by means of x-ray diffraction, in a complex with pol II. We report on the structure of a complete set of GTFs, assembled with pol II and promoter DNA in a 32-protein, 1.5 megaDalton “pre-initiation complex” (PIC), as revealed with cryo-electron microscopy (cryo-EM) and chemical cross-linking. 介绍 RNAⅡ型聚合酶能进行RNA合成但无法识别启动子或启动转录过程。通用转录因子(GTFs)才能实现这些基本核心功能。我们标记这些通用转录因子为TFIIB,-D,-E, -F,和 –H。在补充阶段,这些通用转录因子先是护送启动子DNA到Ⅱ型聚合酶处,接着进行自我展开以便产生出一个转录空泡,然后降落到Ⅱ型聚合酶的裂口处,最后RNA进行合成生成了长度为25的残基且转变成为一条稳定伸长的复合物。我们对有关这一系列变化的结构性基础原理很大程度上仍所知甚少。其中仅有TFIIB的结构基础我们已经通过对含有Ⅱ型聚合酶的复合物进行X射线衍射的方法得以知晓。借助于低温电子显微镜和化学交联技术手段,我们得以确定了整套通用转录因子(GTFs)的完整结构,伴随这套结构所配备的是含有32个蛋白,1.5兆道尔顿前起始复合物(PIC)中含有的Ⅱ型聚合酶和启动子DNA。 Methods Three technical advances enabled the structural analysis of the PIC. First, a procedure was established for the preparation of a stable, abundant PIC. Both the homogeneity and functional activity of the purified PIC were demonstrated. Second, an algorithm was developed for alignment of cryo-EM images that requires no prior information (no “search model”) and that can distinguish multiple conformational states. Last, a computational method was devised for determining the arrangement of protein subunits and domains within a cryo-EM density map from a pattern of chemical cross-linking. 方法 三方面技术上的进步完善使得对前起始复合物的结构性分析变成可能。首先我们为产生稳定丰富的前起始复合物做好了整个准备过程,且检验了已纯化的前起始复合物的一致性和机能活性。其次我们开发出了一种算法使得校整冷冻电镜图像时无需先验信息的获取(无需“搜索模式”)这使得我们可以识别多个构象状态。然后我们还为冷冻电镜密度图中的蛋白质亚基和域的排列分布量身定制了一种计算方法,其中蛋白质亚基和域的冷冻电镜密度图是由化学交联模式获得的。 Results The density map of the PIC showed a pronounced division in two parts, one pol II and the other the GTFs. Promoter DNA followed a straight path, in contact with the GTFs but well separated from pol II, suspended above the active center cleft. Cross-linking and computational analysis led to a most probable arrangement of the GTFs, with IIB at the upstream end of the pol II cleft, followed by IIF, IIE, and IIH. The Ssl2 helicase subunit of IIH was located at the downstream end of the cleft. 结果 前起始复合物(PIC)密度图在两方面表现出了显著的分别,其中一方面是Ⅱ型聚合酶,另一方面是通用转录因子(GTFs)。DNA启动子遵循着笔直的路径轨迹移动,它在很好的避开Ⅱ型聚合酶的同时,又准确的和通用转录因子(GTFs)发生接触,然后在活性中心的裂口前停止了前进的脚步。化学交联和计算分析技术手段使得我们得到通用转录因子(GTFs)的最可行分布成为可能,即让IIB首先分布在Ⅱ型聚合酶裂口的进入端,然后再让IIF,IIE和 IIH的紧随其后按序分布。其中IIH的Ssl2解旋酶亚基则位于该裂口的出口端。 Discussion A principle of the PIC revealed by this work is the interaction of promoter DNA with the GTFs and not with pol II. The GTFs position the DNA above the pol II cleft, but interaction with pol II can only occur after melting of the DNA to enable bending for entry in the cleft. Contact of the DNA with the Ssl2 helicase in the PIC leads to melting (in the presence of adenosine triphosphatase). Cryo-EM by others, based on sequential assembly and analysis of partial complexes rather than of the complete PIC, did not show a separation between pol II and GTFs and revealed direct DNA–pol II interaction. The discrepancy calls attention to a role of the GTFs in preventing direct DNA-polymerase interaction. 讨论 这项研究揭示了前起始复合物(PIC)遵循的活动原理,即它选择性的绕开了Ⅱ型聚合酶而准确和DNA启动子发生相互作用。通用转录因子(GTFs)使得DNA置于Ⅱ型聚合酶裂口上端,但只有在DNA分解后,通用转录因子(GTFs)才能和Ⅱ型聚合酶发生相互作用,从而得以弯曲后得以进入该聚合酶裂口中。在前起始复合物(PIC)处DNA和Ssl2解旋酶的亲密接触使得DNA开始发生分解(即三磷酸腺苷的出现表示DNA开始分解)。他人基于部分复合物而非完整前起始复合物(PIC)的排序分配和分析所做的冷冻电镜没有表现出Ⅱ型聚合酶和通用转录因子(GTFs)的分离,只揭示了DNA和Ⅱ型聚合酶直接的相互作用。这一差异将引起我们关于通用转录因子(GTFs)对防止DNA和聚合酶直接相互作用所起的功能地位的关注。

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