Tat Transactivation

Tat反式激活

• 批准号: 6511440
• 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/6511440
• 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.

转录反式激活器TAT是用于HIV复制的必不可少的调节蛋白，并有助于发病机理。 它通过细胞周期蛋白T1（CYCT1）结合了阳性转录伸长因子B（P-TEFB）。 它们共同结合高亲和力和特异性与反式激活响应（TAR）RNA结构的结合。 然后，P-TEFB中的CDK9然后磷酸化C末端结构域（CTD）或RNA聚合酶II（RNAPII）。 这标志着从启动到HIV转录的伸长的过渡。这些相互作用将非常详细。 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。 这些变化可能影响细胞中P-TEFB的活性和代谢。 将创建主要的负CYCT1蛋白和P-TEFB复合物。 另外，将进一步研究人类Cyct1中的多态性。 他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之间的复合物将经过X射线晶体学和NMR。 目的是获得P-TEFB，TAT和TAR之间的复合物的三级结构。要了解TAT的反式激活，必须检查P-TEFB的作用机理。 除CTD和SPT5以外，不同P-TEFB复合物的底物将在生化和遗传上鉴定。 它们将在P-TEFB向转录复合物的不同模式下进行功能测试。将使用异构的DNA和RNA结合蛋白。 最后，不同的细胞周期蛋白T和K将在小鼠中灭活？ 他们将揭示受感染人类Cyct1和CDK9之间复合物的药理灭活可能是如何有害的。最后，负转录伸长因子（N-TEF）之间的相互作用，其中包括DRB敏感性诱导因子（DSIF）和负伸长因子（NELF）和TAT，将在HIV长期重复（LTR）体外和体内剖析。 将研究焦油和RD中下部茎之间的第一个RNA结合相互作用。 后来将添加DSIF和NELF。 然后，所有这些元素将以精心策划的时间方式组合在一起，这应该揭示Tat反式激活的动力学图片。 体外的发现将与细胞中的染色质免疫沉淀相关。