Structure of the Eukaryote Transcription Apparatus

真核生物转录装置的结构

• 批准号: 8632146
• 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
• 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/8632146
• 关键词: Agreement; Chemicals; Complex; Cryoelectron Microscopy; Crystallography; Data; Disease; Electron Microscopy; Electrons; Eukaryota; General Transcription Factors; Genetic Transcription; Goals; Grant; Health; Holoenzymes; Human; Lead; Malignant Neoplasms; Mass Spectrum Analysis; Mediator of activation protein; Methods; Modification; Movement; Polymerase; Procedures; Process; Proteins; RNA Polymerase II; 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; insight; method development; novel therapeutics; particle; promoter; protein S precursor; protein complex; public health relevance; structural biology; transcription factor; transcription factor TFIIH

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. !

我们当前和拟议的研究重点是 RNA 的结构测定 聚合酶 II (pol II) 转录前起始复合物 (PIC)。我们的重大突破 工作导致了 31 种蛋白质 PIC 的组装和结构测定，能够 TATA-box 启动子处转录起始。我们已经确定了31-的结构 通过冷冻电子显微镜 (cryo-EM) 和交联结合质量进行蛋白质 PIC 光谱测定（XL-MS）。下一个项目期的具体目标是： 1) 延长决议期限 31 蛋白 PIC 的当前结构。冷冻电镜数据将通过直接收集 电子探测器并使用分配权重分布的新方法进行处理 而不是独特的方向，并且避免过度拟合。大型重原子团簇将用于 提高对位速度和精度。 2) 主动结构的确定 启动 PIC。添加 ATP 在 PIC 内打开一个“转录气泡”，使 转录的起始。 3)含有TAF复合物的PIC的结构测定。 PIC 在 14 蛋白 TAF 复合物存在的情况下形成的能够在无 TATA 的情况下启动 发起人。 4) pol II-介体复合物(全酶)的结构测定。 21- 蛋白质介体将调控信息传达给 pol II。通过冷冻电镜和图像进行分析 使用新方法进行处理将比以前的结构产生显着改进。 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。通过修改装配 31 蛋白 PIC 程序中，Mediator 可以掺入化学计量复合物中。 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。已形成复合物 31 蛋白 PIC、TAF 复合物和介体成对组合时，应该是 可以直接将所有三个组件组装到 66 蛋白 PIC 中。通过冷冻电镜分析和 将通过 XL-MS 进行。 ！