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. !

我们目前和拟议的研究集中于RNA的结构确定 聚合酶II（POL II）转录预设复合物（PIC）。我们的重大突破 工作导致了31-蛋白质图片的组装和结构确定，能够 在tata-box启动子上启动转录。我们已经确定了31-的结构 蛋白质PIC通过冷冻电子显微镜（Cryo-EM）和交联与质量结合 光谱法（XL-MS）。下一个项目期间的具体目标是：1）分辨率的扩展 31-蛋白质图片的当前结构。 Cryo-Em数据将通过直接收集 电子探测器并采用新方法进行处理，以分配权重的分布而不是 而不是独特的方向，避免过度拟合。大型沉重的原子簇将用于 提高对齐的速度和精度。 2）积极的结构确定 启动图片。 ATP的添加在图片中打开“转录气泡”，并启用 转录的开始。 3）包含TAF复合物的PIC的结构确定。图片 在14-蛋白质TAF复合物存在下形成的能够在tata-noth 发起人。 4）POL II-介体复合物（Holoenzyme）的结构测定。 21- 蛋白质介质将调节信息传达给Pol II。通过冷冻EM和图像分析 使用新方法处理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-介体复合物的结构测定。通过修改组装 31-蛋白质PIC的过程，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） 组装和结构确定完整的66-蛋白质图片。已经形成复合物 在成对组合中的31-蛋白质PIC，TAF复合物和调解人中，应该是 直接在66蛋白图片中组装所有三个组件。 Cryo-Em和 由XL-MS进行。 呢