Structure of the Eukaryote Transcription Apparatus

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）转录起始前复合物（PIC）的结构确定。我们在工作中取得了重大突破，确定了一个具有31个蛋白的PIC的组装和结构，该PIC能够在TATA-box启动子处启动转录。我们用低温电镜（cryo-EM）和交联质谱（XL-MS）测定了31个蛋白的PIC的结构。下一个项目周期的具体目标是：1)扩展31蛋白PIC当前结构的分辨率。Cryo-EM数据将由直接电子探测器收集，并采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。大型重原子团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。ATP的加入在PIC中打开了一个“转录泡”，并使转录开始。3)含TAF配合物的PIC结构测定。在14蛋白TAF复合物存在下形成的PIC能够在无TAF启动子处起始。4) pol ii -中介复合物（全酶）的结构测定。21蛋白中介体向pol II传递调控信息。低温电镜分析和图像处理的新方法将导致比以前的结构显著改善。xml - ms分析将揭示负责转录调控的相互作用。5) PIC-Mediator复合物的结构测定。通过修改31蛋白PIC的组装程序，Mediator可以加入到一个化学计量络合物中。初步的低温电镜和单颗粒分析证实了材料的均匀性。6)完整的66蛋白PIC的组装和结构测定。在形成了31蛋白的PIC、TAF复合物和Mediator的成对组合后，将这三种成分组装成66蛋白的PIC应该是很简单的。将进行低温电镜和XL-MS分析。

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.