HUMAN T CELLS

人类T细胞

• 批准号: 2542416
• 负责人: Ajit Kumar
• 金额: $ 7.9万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2542416
• 关键词: DNA directed RNA polymerase; T lymphocyte; complementary DNA; enzyme activity; genetic library; human immunodeficiency virus 1; immunoprecipitation; phosphoprotein phosphatase; phosphorylases; protein purification; recombinant proteins; tissue /cell culture; transcription factor; virus infection mechanism; virus protein

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.

描述（改编自申请人摘要）：人类免疫缺陷 1型病毒（HIV-1）调节蛋白达特增加了全 从病毒LTR的长度RNA转录本。 达特与转录因子相互作用 因子和RNA聚合酶II（RNAP II）酶复合物，以增加 转录延长过程的效率。 一种可能的机制， 达特功能提出了达特与特定运动的关联， 磷酸化RNAPII大亚基的C末端结构域（CTD）。 低磷酸化的RNAPII与转录相关， 起始复合物，而过度磷酸化的RNAPII与 延伸复合体和低磷酸化形式的再生 下一轮转录需要用到 我们推断， 达特相关的磷酸酶可能与达特和RNAPII相互作用， 转录复合物以再生RNAPII的低磷酸化形式。 我们已经部分表征了达特相关磷酸酶， 对冈田酸敏感，并通过达特 相关CTD激酶（TTK）。 该项目的长期目标是 从人T细胞中分离编码达特相关磷酸酶的cDNA 并对其在达特反式激活中的作用机制进行了初步探讨。 达特相关磷酸酶的底物特异性将是 通过使用各种磷酸化蛋白质如大的 RNA聚合酶II亚基，RNAP II的重组C-末端结构域，dk 7， 和磷酸化酶A。达特的结构域要求和 将通过使用达特突变体来研究磷酸酶和达特相互作用 和一个无关的病毒反式激活因子VP 16。 在生协会 将通过免疫沉淀达特来研究磷酸酶与达特的关系 来自HeLa细胞系的细胞裂解物的相关蛋白质表达 HA标记的达特蛋白。 然后，将达特相关磷酸酶 通过色谱法纯化。 抗体将使用 所述寡肽对纯化的磷酸酶具有特异性。 的cDNA 将通过筛选来自T. 细胞及其特性和功能。 这些研究将 不仅有助于更好地了解达特的机制， 响应，他们针对与RNA的基本机制相关的问题 转录。