Structure of the Eukaryote Transcription Apparatus

描述(申请人提供)：我们目前和未来的研究重点是RNA聚合酶II(PolII)转录起始前复合体(PIC)的结构确定。我们工作中的一项重大突破导致了31蛋白PIC的组装和结构测定，该PIC能够在TATA-box启动子上启动转录。我们用低温电子显微镜(Cryo-EM)和交联质谱(XL-MS)确定了31-蛋白PIC的结构。下一个项目期的具体目标是：1)延长对31蛋白PIC当前结构的解析。CRYO-EM数据将用直接电子探测器收集，并用新的方法处理，这种方法分配权重分布，而不是唯一的方向，避免过度拟合。大型重原子团簇将被用来提高对准的速度和精度。2)主动引发PIC的结构确定。三磷酸腺苷的加入打开了PIC内的“转录泡泡”，使转录的启动成为可能。3)含TAF络合物的PIC的结构测定。在14蛋白TAF复合体存在下形成的PIC能够在没有TATA的启动子上启动。4)PolII-介体复合体(全酶)的结构测定。21蛋白介体向POLII传达调控信息。低温EM分析和新方法的图像处理将导致对以前结构的显著改进。XL-MS分析将揭示与转录调控有关的相互作用。5)PIC-介体复合体的结构确定。通过修改31蛋白PIC的组装程序，Mediator可以被整合到化学计量复合体中。初步的低温电子显微镜和单粒子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PIC、TAF复合体和介体的两两结合的复合体后，将所有这三个组分组装成66蛋白PIC应该是简单的。将进行冷冻-EM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.