Structure of the Eukaryote Transcription Apparatus

真核生物转录装置的结构

基本信息

批准号：
9189671
负责人：
ROGER D KORNBERG
金额：
$ 60.99万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-05-01 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9189671
关键词：
Agreement Chemicals Complex Cryoelectron Microscopy Crystallography DNA Polymerase II DNA-Directed RNA Polymerase Data Disease Electron Microscopy Electrons Eukaryota Genetic Transcription Goals Grant Health Holoenzymes Human Malignant Neoplasms Mass Spectrum Analysis Mediator of activation protein Methods Modification Movement Procedures Process Proteins Regulation Research Resolution Specimen Speed Structure TATA Box Transcription Initiation Transcriptional Regulation Weight Work X ray diffraction analysis X-Ray Diffraction Yeasts crosslink detector electron tomography image processing imaging modality insight method development novel therapeutics particle pol Gene Products promoter protein S precursor protein complex public health relevance structural biology transcription factor transcription factor S-II transcription factor TFIIH

项目摘要

DESCRIPTION (provided by applicant): Our current and proposed research is focused on structure determination of the RNA polymerase II (pol II) transcription pre-initiation complex (PIC). A major breakthrough in our work has led to the assembly and structure determination of a 31-protein PIC, capable of the initiation of transcription at TATA-box promoters. We have determined the structure of the 31-protein PIC by cryo-electron microscopy (cryo-EM) and cross-linking combined with mass spectrometry (XL-MS). Specific aims for the next project period are: 1) Extension of resolution of the current structure of the 31-protein PIC. Cryo-EM data will be collected with a direct electron detector and processed with new methods that assign distributions of weights rather than unique orientations, and that avoid overfitting. Large heavy atom clusters will be used to enhance the speed and precision of alignment. 2) Structure determination of an actively initiating PIC. Addition of ATP opens a "transcription bubble" within the PIC and enables the initiation of transcription. 3) Structure determination of PIC containing the TAF complex. PIC formed in the presence of the 14-protein TAF complex is capable of initiation at TATA-less promoters. 4) Structure determination of pol II-Mediator complex (holoenzyme). The 21-protein Mediator communicates regulatory information to pol II. Analysis by cryo-EM and image processing with new methods will result in significant improvement over previous structures. Analysis by XL-MS will reveal interactions responsible for the regulation of transcription. 5) Structure determination of a PIC-Mediator complex. By modification of the assembly procedure for the 31-protein PIC, Mediator could be incorporated in a stoichiometric complex. Preliminary cryo-EM and single particle analysis has confirmed the uniformity of the material. 6) Assembly and structure determination of a complete 66-protein PIC. Having formed complexes of the 31-protein PIC, TAF complex, and Mediator in pairwise combinations, it should be straightforward to assemble all three components in a 66-protein PIC. Analysis by cryo-EM and by XL-MS will be undertaken.

描述（由申请人提供）：我们当前和拟议的研究重点是 RNA 聚合酶 II (pol II) 转录前起始复合物 (PIC) 的结构测定。我们的工作取得了重大突破，实现了 31 种蛋白质 PIC 的组装和结构测定，该 PIC 能够在 TATA-box 启动子处启动转录。我们通过冷冻电子显微镜 (cryo-EM) 和交联结合质谱 (XL-MS) 确定了 31 种蛋白质 PIC 的结构。下一个项目期的具体目标是： 1) 扩展 31 蛋白 PIC 当前结构的分辨率。冷冻电镜数据将通过直接电子探测器收集，并使用分配权重分布而不是唯一方向的新方法进行处理，并避免过度拟合。大型重原子簇将用于提高对准的速度和精度。 2)主动启动PIC的结构确定。添加 ATP 在 PIC 内打开一个“转录泡”并启动转录。 3)含有TAF复合物的PIC的结构测定。在 14 蛋白 TAF 复合物存在的情况下形成的 PIC 能够在无 TATA 的启动子处启动。 4) pol II-Mediator复合物(全酶)的结构测定。 21 蛋白介体将调控信息传达给 pol II。通过冷冻电镜分析和新方法的图像处理将比以前的结构产生显着的改进。 XL-MS 分析将揭示负责转录调节的相互作用。 5) PIC-介体复合物的结构测定。通过修改 31 蛋白 PIC 的组装程序，Mediator 可以整合到化学计量复合物中。初步冷冻电镜和单颗粒分析已证实材料的均匀性。 6) 完整的 66 蛋白 PIC 的组装和结构测定。在成对组合形成 31 蛋白 PIC、TAF 复合物和介体的复合物后，将所有三个组件组装到 66 蛋白 PIC 中应该很简单。将通过冷冻电镜和 XL-MS 进行分析。