Structure of the Eukaryote Transcription Apparatus

真核生物转录装置的结构

• 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
• 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

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集中于RNA聚合酶II（POL II）转录预发射复合物（PIC）的结构确定。我们工作的一个重大突破导致了31-蛋白质PIC的组装和结构测定，该图片能够在Tata-box启动子上启动转录。我们通过冷冻电子显微镜（Cryo-EM）确定了31-蛋白质PIC的结构，并与质谱（XL-MS）结合了交联。下一个项目期间的具体目的是：1）扩展31-蛋白质PIC的当前结构的分辨率。冷冻EM数据将使用直接电子检测器收集，并使用分配权重而不是唯一方向的新方法处理，并避免过度拟合。大型重原子簇将用于提高对齐的速度和精度。 2）积极启动图片的结构确定。 ATP的添加在PIC中打开“转录气泡”，并可以启动转录。 3）包含TAF复合物的PIC的结构确定。在14-蛋白质TAF复合物存在下形成的PIC能够在无TATA的启动子处启动。 4）POL II-MEDIARITER复合物（Holoenzyme）的结构测定。 21-蛋白质介质将监管信息传达给Pol II。通过冷冻EM和图像处理的分析新方法将导致对先前结构的显着改善。 XL-MS的分析将揭示负责转录调节的相互作用。 5）PIC-MEDIARITER复合物的结构测定。通过修改31-蛋白质PIC的组装过程，可以将介体纳入化学计量学复合体中。初步的冷冻EM和单个颗粒分析已经证实了材料的均匀性。 6）完整的66蛋白图片的组装和结构测定。在成对组合中形成了31-蛋白质PIC，TAF复合物和介体的复合物，应该直接在66-蛋白质PIC中组装所有三个组件。将进行Cryo-EM和XL-MS的分析。