STRUCTURE OF RNA POLYMERASE II

我们研究的长期目标是阐明这一机制和 真核生物转录调控。我们关注的是RNA聚合酶II， 负责几乎所有真核转录的酶，其 错综复杂的调控是许多细胞和发育过程的基础。 大多数调控发生在转录的起始阶段，这涉及到 RNA聚合酶II与一组通用转录因子的相互作用 推动者的因素。RNA聚合酶II本身就是一个大的组装： 酵母酶由15条多肽链组成，其质量接近 60万道尔顿。引发复合体由25个以上的 多肽，总质量超过120万道尔顿。我们希望 来确定整个起爆的三维结构 复杂，作为理解机制和 转录调控。我们将零碎地解决结构：X- 聚合酶和一般转录因子的射线结构将是 单独确定，这些结构将组合在 聚合酶因子复合体的电子结晶学基础。我们 提出了最近对RNA聚合酶II晶体进行的X射线分析 并对聚合酶共晶的形成和分析进行了研究 DNA、RNA和一般转录因子。结果应该适用于 RNA聚合酶和转录机制，因为 聚合酶、转录因子和机制方面的研究 转录调控在进化过程中是保守的。目标 而拟议研究的实验方法包括 以下是： 1.酵母菌RNA聚合酶II的X射线结构将在 分辨率低(6-12埃)。来自电子结晶学的信息 将被用来代替重原子衍生物中的相。这个 成果将有助于实现以下所有进一步目标 这个项目。 2.酵母RNA聚合酶II的X射线结构将在 原子分辨率。将使用重原子簇合物来制备 衍生晶体，重原子的位置将会被找到 在低分辨率聚合酶结构的帮助下。 3.RNA聚合酶II与DNA和RNA寡核苷酸的共晶将 用差分傅立叶方法进行制备和分析。初步分析 在低分辨率下将足以显示模板和产品 酶的结合部位。“伸长”复合体，含有这种酶 在转录行为中，也将被结晶并受制于 结构确定。 4.RNA聚合酶II与RPB4和RPB4亚基的复合体的晶体 将制备具有通用转录因子e(TFIIB)的RPB7。 同样，在低分辨率下的初步分析将足以揭示 与聚合酶的相互作用。

The long term goal of our research is to elucidate the mechanism and regulation of transcription in eukaryotes. We focus on RNA polymerase II, the enzyme responsible for almost all eukaryotic transcription, whose intricate regulation underlies many cellular and developmental processes. Most regulation occurs at the initiation of transcription, which involves the interaction of RNA polymerase II with a set of general transcription factors at a promoter. RNA polymerase II is itself a large assembly: the yeast enzyme is made up of 15 polypeptide chains, with a mass of nearly 600,000 Daltons. The initiation complex comprises more than 25 polypeptides, with a total mass in excess of 1.2 million Daltons. We wish to determine the three-dimensional structure of the entire initiation complex, as an essential step towards understanding the mechanism and regulation of transcription. We will solve the structure piecemeal: the X- ray structures of the polymerase and general transcription factors will be determined individually, and these structures will be combined on the basis of electron crystallography of polymerase-factor complexes. We propose here the X-ray analysis of RNA polymerase II crystals recently obtained, and the formation and analysis of cocrystals of the polymerase with DNA, RNA, and general transcription factors. The results should apply to RNA polymerases and transcription mechanisms in general, since the polymerases, transcription factors, and aspects of the mechanism and regulation of transcription have been conserved through evolution. Aims and experimental approaches of the proposed research include the following: 1. The X-ray structure of yeast RNA polymerase II will be determined at low (6-12 Angstroms) resolution. Information from electron crystallography will be employed in lieu of phases from heavy atom derivatives. The results will be instrumental in achieving all the further objectives of the project. 2. The X-ray structure of yeast RNA polymerase II will be determined at atomic resolution. Heavy atom cluster compounds will be used to prepare derivative crystals, and the locations of the heavy atoms will be found with the aid of the low resolution polymerase structure. 3. Cocrystals of RNA polymerase II with DNA and RNA oligonucleotides will be prepared and analyzed by difference Fourier methods. Analysis initially at low resolution will be sufficient to reveal the template and product binding sites of the enzyme. "Elongation" complexes, containing the enzyme in the act of transcription, will also be crystallized and subjected to structure determination. 4. Cocrystals of RNA polymerase II with a complex of subunits RPB4 and RPB7 and with general transcription factor e (TFIIB) will be prepared. Again initial analysis at low resolution will suffice to reveal sites of interaction with the polymerase.