RNA POLYMERASE II ELONGATION COMPLEX--STRUCTURE/FUNCTION

描述(部分改编自申请者的描述)：本项目 研究TFIIS(SII)转录本切割因子的作用机制 以及它在转录调控中的作用。SII通过以下方式刺激转录 与暂停或停滞的RNA聚合酶II结合并激活潜伏期 酶中的核酸酶，该酶将新生的转录本在其3‘端附近裂解为 实现各种数据块的通读，以链延长。这个项目将 继续探索SII的机制，重点放在它与 新生的核糖核酸。RNA在SII上的接触部位将被精确绘制。突变型 消除RNA接触的SII蛋白将被产生并在体外进行测试 SII基因缺失的活性和补充表型的能力 酵母。聚合酶上的SII接触点将由蛋白酶绘制 脚印。核染色质上RNA聚合酶II受阻的直接证据 将使用核奔跑和染色质免疫沉淀来寻找活体 用抗聚合酶抗体进行检测。碱基含量有偏差的基因将是 调查以了解NTP池是否调节基因特异性基因的延伸效率 在活体内的方式。另一个目的是确定用于调节PUR5的机制 在酵母中表达对NTP池减少的响应。这将包括 在不同生长条件下测量NTP池并响应 6-氮卓酮，一种UTP和GTP合成的抑制剂。最后，老鼠被扰乱了 将生成两个SII家族基因(Tcea1和Tcea3)并对其进行分析 发育表型。

DESCRIPTION (adapted in part from applicant's description): This project examines the mechanism of action of the TFIIS (SII) transcript cleavage factor and its role in transcriptional regulation. SII stimulates transcription by binding to paused or arrested RNA polymerase II and activating a latent nuclease in the enzyme that cleaves the nascent transcript near its 3' end to enable readthrough of various blocks to chain elongation. This project will continue to explore SII's mechanism with a focus on its interaction with nascent RNA. The contact site of RNA on SII will be precisely mapped. Mutant SII proteins eliminating RNA contact will be generated and tested for in vitro activity and an ability to complement the phenotypes of an SII gene deletion in yeast. SII contact sites on the polymerase will be mapped by protease footprinting. Direct evidence for arrested RNA polymerase II on chromatin in vivo will be sought using nuclear run-on and chromatin immunoprecipitation assays with anti-polymerase antibodies. Genes with biased base content will be surveyed to see if NTP pools regulate elongation efficiency in a gene-specific manner in vivo. Another aim is to determine the mechanism used to regulate PUR5 expression in response to NTP pool reduction in yeast. This will include measurement of NTP pools under various growth conditions and in response to 6-azauracil, an inhibitor of UTP and GTP synthesis. Finally, mice disrupted for two SII family genes (TceaI and Tcea3) will be generated and analyzed for developmental phenotypes.