STRUCTURE OF RNA POLYMERASE II

这项拟议研究的目标是在原子分辨率下确定RNA聚合酶II及其与核酸和辅助蛋白质因子的络合物的X射线结构。这个问题很有挑战性，因为仅聚合酶就由15个多肽组成，总质量近60万道尔顿，加上辅助因子，多肽的数量和蛋白质质量都翻了一番。下一个项目的具体目标如下：1.原子分辨下的RNA聚合酶II的X射线结构测定。该酶的两种新晶型，衍射率分别为3.1和2.9奥格斯特龙单位，以及目前已有的高于4奥格斯特罗姆单位分辨率的相，有望早日实现这一目标。将采取几种方法，包括收集额外的导数数据、晶体平均和改进现有数据的处理。2.RNA聚合酶II与DNA、RNA、DNA和RNA(延伸复合体)、TFIIB、TFIIE、TFIIS、Rpb4、Rpb7和-Amanitin的原子分辨X射线结构测定。从九种晶体类型中的五种获得了数据集，这些晶体具有可接受的加工统计数据，分辨率达到3.5奥格斯特龙单位或更高。将寻求分子替代解决方案，或将制备衍生品，并使用先前使用的衍生品收集X射线数据。3.抑制RNA聚合酶II转录复合体的结晶和结构测定。最近对大肠杆菌RNA聚合酶延长复合体的研究表明，该酶在转录活性和非活性状态之间振荡，分别有助于RNA链的延长和RNA的切割/校对。我们目前对暂停的(活跃的)RNA聚合酶II延伸复合体的结晶学研究将扩展到停滞的(不活跃的)复合体。4.庞氏酵母和人RNA聚合酶的结晶。初步结果表明，将我们的研究扩展到这些重要的酶是可行的。它们之间的亲缘关系可能比我们目前研究的对象酿酒酵母的酶更密切。

The goal of the proposed research is to determine the X-ray structures of RNA polymerase II and of its complexes with nucleic acids and auxiliary protein factors at atomic resolution. The problem is challenging, since the polymerase alone comprises 15 polypeptides with a total mass of nearly 600,000 Daltons, and addition of the auxiliary factors doubles both the number of polypeptides and the protein mass. Specific aims for the coming project period are as follows: 1. X-ray structure determination of RNA polymerase II at atomic resolution. Two new crystal forms of the enzyme, diffracting to 3.1 and 2.9 Augstrom units resolution, as well as a phase set at better than 4 Augstrom units resolution currently in hand, promise the early accomplishment of this aim. Several approaches will be taken, including the collection of additional derivative data, crystal averaging, and improved processing of existing data. 2. X-ray structure determination of RNA polymerase II complexed with DNA, RNA, DNA and RNA (elongation complex), TFIIB, TFIIE, TFIIS, Rpb4, Rpb7, and -amanitin at atomic-resolution. Datasets have been obtained from five of the nine crystal types with acceptable processing statistics to 3.5 Augstrom units resolution or better. Molecular replacement solutions will be sought, or derivatives will be prepared and X-ray data collected with the use of the derivatives previously employed. 3. Crystallization and structure determination of an arrested RNA polymerase II transcription complex. Recent studies of E. coli RNA polymerase elongation complexes have revealed an oscillation of the enzyme between transcriptionally active and inactive states, conductive to RNA chain elongation and RNA cleavage/proofreading respectively. Our current crystallographic studies of a paused (active) RNA polymerase II elongation complex will be extended to an arrested (inactive) complex. 4. Crystallization of Scizosaccharomyces pombe and human RNA polymerases. Preliminary results suggest the feasibility of extending our studies to these important enzymes. They are likely to be more closely related to one another than to the enzyme from Saccharomyces cerevisiae, the object of our current investigations.