Tat Transactivation

Tat反式激活

• 批准号: 6603278
• 负责人: Boris Matija PETERLIN
• 金额: $ 33.19万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6603278
• 关键词: 3T3 cells; AIDS; DNA binding protein; HeLa cells; RNA binding protein; SDS polyacrylamide gel electrophoresis; Sf9 cell line; X ray crystallography; autoradiography; clone cells; gene induction /repression; gene targeting; genetic mapping; genetic polymorphism; human immunodeficiency virus; immunoprecipitation; laboratory mouse; nuclear magnetic resonance spectroscopy; nucleic acid sequence; phosphorylation; polymerase chain reaction; posttranscriptional RNA processing; protein structure function; thin layer chromatography; transcription factor; translation factor; virus genetics; virus replication; western blottings

The transcriptional transactivator Tat is an essential regulatory protein for HIV replication and AIDS pathogenesis. It binds to the positive transcription elongation factor b (P-TEFb) via the cyclin T1 (cycT1). Together, they bind with high affinity and specificity to the transactivation response (TAR) RNA structure. Cdk9 in P-TEFb then phosphorylates the C-terminal domain (CTD) or RNA polymerase II (RNAPII). This marks the transition from initiation to elongation of HIV transcription. These interactions will be dissected in great detail. Extensive mutageneses will reveal precise binding surfaces on Tat and cycT1. Posttranscriptional modification of cycT1 will be analyzed. These changes could affect the activity and metabolism of P-TEFb in cells. Dominant negative cycT1 proteins and P-TEFb complexes will be created. Additionally, polymorphisms in the human cycT1 will be examined further. They could play an important role in the host response to HIV. The complex between P-TEFb, Tat and TAR will be subjected to X-ray crystallography and NMR. The goal is to obtain the tertiary structure of the complex between P-TEFb, Tat and TAR. To understand Tat transactivation, the mechanism of action of P- TEFb must be examined. Substrates of different P-TEFb complexes besides CTD and SPT5 will be identified biochemically and genetically. They will be tested functionally on different modes of presentation of P-TEFb to the transcription complex. Heterogous DNA and RNA binding proteins will be used. Finally, different cyclins T and K will be inactivated genetically in the mouse? They will reveal how deleterious might be the pharmacological inactivation of the complex between cycT1 and Cdk9 in infected humans. Finally, the interplay between the negative transcription elongation factor (N-TEF), which includes the DRB-sensitivity inducing factor (DSIF) and negative elongation factor (NELF) and Tat will be dissected on the HIV long terminal repeat (LTR) in vitro and in vivo. First RNA-binding interactions between the lower stem in TAR and RD will be studied. Later DSIF and NELF will be added. Then, all these elements will be combined in a carefully orchestrated temporal fashion, which should reveal the kinetic picture of Tat transactivation. Findings in vitro will be correlated with chromatin immunoprecipitations in cells.

转录反式激活蛋白 Tat 是 HIV 复制和 AIDS 发病机制的重要调节蛋白。 它通过细胞周期蛋白 T1 (cycT1) 与正转录延伸因子 b (P-TEFb) 结合。 它们一起以高亲和力和特异性结合反式激活反应 (TAR) RNA 结构。 P-TEFb 中的 Cdk9 随后磷酸化 C 末端结构域 (CTD) 或 RNA 聚合酶 II (RNAPII)。 这标志着HIV转录从起始到延长的转变。这些相互作用将被详细剖析。 广泛的诱变将揭示 Tat 和 cycT1 上的精确结合表面。 将分析 cycT1 的转录后修饰。 这些变化可能会影响细胞中 P-TEFb 的活性和代谢。 将产生显性负性 cycT1 蛋白和 P-TEFb 复合物。 此外，人类 cycT1 的多态性将得到进一步检查。 它们可以在宿主对艾滋病毒的反应中发挥重要作用。 P-TEFb、Tat 和 TAR 之间的复合物将进行 X 射线晶体学和 NMR 分析。 目标是获得 P-TEFb、Tat 和 TAR 之间复合物的三级结构。为了了解 Tat 反式激活，必须检查 P-TEFb 的作用机制。 除了 CTD 和 SPT5 之外，还将通过生化和遗传学方法鉴定不同 P-TEFb 复合物的底物。 他们将在 P-TEFb 向转录复合物呈递的不同模式上进行功能测试。将使用异源 DNA 和 RNA 结合蛋白。 最后，不同的细胞周期蛋白T和K会在小鼠体内基因失活吗？ 他们将揭示 cycT1 和 Cdk9 之间的复合物在受感染的人类体内的药理失活可能是多么有害。最后，负转录延伸因子 (N-TEF)（包括 DRB 敏感性诱导因子 (DSIF) 和负延伸因子 (NELF)）与 Tat 之间的相互作用将在体外在 HIV 长末端重复序列 (LTR) 上进行剖析和体内。 首先将研究 TAR 和 RD 下茎之间的 RNA 结合相互作用。 稍后将添加 DSIF 和 NELF。 然后，所有这些元素将以精心策划的时间方式组合起来，这应该揭示 Tat 反式激活的动力学图景。 体外研究结果将与细胞中染色质免疫沉淀相关。