HUMAN T CELLS

描述(摘自申请者摘要)：人类免疫缺陷 1型病毒(HIV-1)调节蛋白TAT增加完整的合成 来自病毒LTR的长度RNA转录本。TAT与转录子相互作用 因子和RNA聚合酶II(RNAP II)酶复合体增加 记录延长过程的效率。一种可能的机制是 TAT功能建议将TAT与特定的运动相关联，该运动 使RNAPII大亚基的c-末端结构域(CTD)磷酸化。 低磷酸化的RNAPII与转录相关 起始复合体，而过度磷酸化的RNAPII与 延伸复合体和低磷酸化形式的再生 将是下一轮转录所必需的。我们推断出一个 TAT相关磷酸酶可能与TAT和RNAPII相互作用 转录复合体，以再生低磷酸化形式的RNAPII。 我们已经部分鉴定了一种与TAT相关的磷酸酶 对冈田酸敏感，使CTD磷酸化降低一倍 相关CTD激酶(TTK)。这个项目的长期目标是 从人T细胞中分离编码TAT相关磷酸酶的基因 并对其在TAT反式激活中的作用机制进行了表征。 TAT相关磷酸酶的底物专一性将是 通过使用各种磷酸化的蛋白质来确定，例如大的 RNA聚合酶II的亚单位，重组的RNAP II的C末端结构域，dk7， 和磷酸化酶a.TAT的结构域要求和TAT的特异性 磷酸酶和tat的相互作用将利用tat的突变体进行研究。 和一个无关的病毒反式激活剂VP16。在万岁协会中 具有TAT的磷酸酶将通过免疫沉淀TAT来研究 HeLa细胞系细胞裂解液中相关蛋白的表达 HA标记的TAT蛋白。之后，与TAT相关的磷酸酶将被 通过层析提纯。将使用以下方法生成抗体 纯化的磷酸酶专一性的寡肽。编码该基因的c DNA 从T细胞中筛选出一个表达文库，分离出磷酸酶 细胞及其特性和功能的表征。这些研究将 不仅有助于更好地了解TAT的机制 作为回应，它们针对与RNA的基本机制相关的问题 抄写。

DESCRIPTION (adapted from applicant's abstract): Human Immunodeficiency virus type 1 (HIV-1) regulatory protein Tat increases the synthesis of full length RNA transcripts from the viral LTR. Tat interacts with transcripion factors and RNA polymerase II (RNAP II) enzyme complex to increase the efficiency of the transcript elongation process. One possible mechanism for Tat function proposes the association of Tat with specific kineses which phosphorylate the c-terminal domain (CTD) of the large subunit of RNAPII. The hypophosphorylated RNAPII is associated with the transcription initiation complex while the hyperphosphorylated RNAPII is associated with the elongation complex, and the regeneration of the hypophosphorylated form will be required for the next round of transcription. We reasoned that a Tat associated phosphatase may interact with Tat and RNAPII in the transcription complex to regenerate the hypophosphorylated form of RNAPII. We have partially characterized a Tat associated phosphatase which is sensitive to okadaic acid and decreases the CTD phosphorylation by a Tat associated CTD kinase (TTK). The long term goal of this project is to isolate the cDNA encoding the Tat associated phosphatase from human T cells and characterize the mechanism of its function in Tat trans-activation. The substrate specificity of the Tat associated phosphatase will be determined by using various phosphorylated proteins such as the large subunit of RNA polymerase II, recombinant c-terminal domain of RNAP II, dk7, and phosphorylase a. The domain requirement of Tat and the specificity of the phosphatase and Tat interaction will be studied by using mutants of Tat and an unrelated viral transactivator VP16. In viva association of the phosphatase with Tat will be studied by immunoprecipitating the Tat associated proteins from the cell Iysate of a HeLa cell line expressing HA-tagged Tat protein. Afterwards, the Tat associated phosphatase will be purified by chromatography. Antibodies will be generated using oligopeptides sepecific to the purified phosphatase. The cDNA encoding the phosphatase will be isolated by screening a cDNA expression library from T cells and its properties and functions characterized. These studies will not only help in gaining a better understanding of the mechanism of Tat response, they target issues relevant to basic mechanisms of RNA transcription.