Structure of the Eukaryote Transcription Apparatus

真核生物转录装置的结构

• 批准号: 9011984
• 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/9011984
• 关键词: Agreement; Chemicals; Complex; Cryoelectron Microscopy; Crystallography; Data; Disease; Electron Microscopy; Electrons; Eukaryota; 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; microscopic imaging; novel therapeutics; particle; promoter; protein S precursor; protein complex; structural biology; transcription factor; transcription factor TFIIH

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）：我们目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。在我们的工作中的一个重大突破，导致了31蛋白质PIC的组装和结构确定，能够启动转录TATA盒启动子。我们已经确定了结构的31蛋白质PIC的冷冻电子显微镜（cryo-EM）和交联结合质谱（XL-MS）。下一个项目周期的具体目标是：1）延长31蛋白PIC当前结构的分辨率。将使用直接电子探测器收集Cryo-EM数据，并使用分配权重分布而不是唯一方向的新方法进行处理，并避免过拟合。大的重原子团簇将用于提高对准的速度和精度。2)活性起始PIC的结构测定。ATP的加入打开PIC内的“转录泡”，并使转录启动。3)含有TAF复合物的PIC的结构测定。在14-蛋白TAF复合物存在下形成的PIC能够在TATA较少的启动子处起始。4)pol II-介体复合物（全酶）的结构测定。21-蛋白质介体将调节信息传递给pol II。通过冷冻EM和图像处理与新方法的分析将导致显着改善以前的结构。通过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-蛋白质PIC的组装程序，可以将介体掺入化学计量复合物中。初步的冷冻EM和单颗粒分析证实了材料的均匀性。6)一个完整的66蛋白质PIC的组装和结构测定。在形成了31-蛋白质PIC、TAF复合物和介体成对组合的复合物之后，将所有三种组分组装成66-蛋白质PIC应该是简单的。将通过冷冻EM和XL-MS进行分析。