TAT Transactivation

TAT反式激活

基本信息

批准号：
8138042
负责人：
Boris Matija PETERLIN
金额：
$ 23.18万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-27 至 2011-09-26
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8138042
关键词：
Acquired Immunodeficiency Syndrome Affinity Antibodies Applications Grants B-Lymphocytes Binding Biological Assay C-terminal CDK9 Protein Kinase Cell physiology Cells Code Complex Cyclins DNA Dactinomycin Elongation Factor Enhancers Equilibrium Future Genes Genetic Genetic Transcription Genome HIV HIV Enhancer HIV-1 Hexamethylene Bisacetamide Human Immunoglobulin binding proteins In Vitro Knock-out Knowledge Lead Long Terminal Repeats Maps Messenger RNA Modification Molecular Conformation Molecular Weight Mus NF-kappa B Nuclear Organism Phosphorylation Phosphotransferases Play Post-Translational Protein Processing Process Proteins RNA RNA Polymerase II RNA-Binding Proteins RNA-Protein Interaction Recruitment Activity Regulation Research Personnel Role Small Nuclear RNA Solutions Specificity Stimulus Stress Structure Surface TNFRSF5 gene Therapeutic Intervention Trans-Activators Transactivation Transcript Transcription Elongation Ubiquitin Ultraviolet Rays Viral X-Ray Crystallography base beta-microseminoprotein cDNA Arrays cell type cellular targeting chromatin immunoprecipitation combinatorial cyclin T1 in vivo inhibitor/antagonist interest kappa-Chain Immunoglobulins mutant negative elongation factor p65 programs protein complex reconstitution response stoichiometry three dimensional structure

项目摘要

The long term objectives of this project are to understand how the process of elongation of transcription is regulated in cells and on the Human Immunodeficiency Virus type 1 Long Terminal Repeat (HIVLTR). The general strategy is to identify roles played by different Positive Transcription Elongation Factor b (P- TEFb) complexes, HEXamethylene blsacetiMide-inducible proteins 1 and 2 (HEXIM1/2), 7SK small nuclear RNA (7SK RNA), nuclear factor kappa B (NF-KB), HIV transcriptional Transactivator (Tat) and TransActivation Response (TAR) RNA in this process. P-TEFb contains the Cyclin dependent kinase 9 (Cdk9) and a C-type cyclin, CycT1, CycT2 or CycK. NF-KB contains p65 (RelA) and p50. Of special importance is the balance between inactive, high molecular weight (HMW) P-TEFb complexes that contain additionally 7SK RNA and HEXIM1/2 and active, low molecular weight (LMW) P-TEFb complexes that contain only Cdk9 and a C-type cyclin. The specific aims are to define as precisely as possible surfaces that form inactive, HMW P-TEFb and active, LMW P-TEFb complexes that bind NF-KB and Tat. Two conformational states of HEXIM1/2 exist in cells, both oligomeric, one complexed with 7SK RNA and P-TEFb, the other free in solution. In addition, we want to determine the precise three-dimensional structure of the complex between CycT1, Tat and TAR, with special emphasis on surfaces on these proteins that bind RNA and the now stabilized TAR. Next, we shall investigate the function of these different P-TEFb complexes and how they are regulated by posttranslational modifications. Of special interest is the phosphorylation and ubiquitylation of HEXIM1/2. Once activated, how is P-TEFb recruited by NF-KB to the HIVLTR? What role does the ubiquitylation of p65 play in this process? From other studies, we know that monoubiquitin binds the C-terminus of CycT1. These studies will lead to the creation of constitutively active HEXIM1/2 proteins, which will be used to determine their differential effects on cellular and viral transcription. Finally, we shall compare the recruitment of P-TEFb by NF-KB via DNA and Tat via RNA in vitro and in vivo, study the dynamic interplay between Negative Elongation Factor (N-TEF) and P-TEFb on the HIVLTR by chromatin immunoprecipitation in cells as well as inactivate genes encoding CycT1, CycT2 and CycK in the mouse. Unique and redundant roles of P-TEFb complexes containing these C-type cyclins will be revealed in the organism. These studies will reveal the complex RNA-protein world of P-TEFb and how HIV subverts these cellular complexes for its replication in cells.

该项目的长期目标是了解转录的伸长过程是如何的在细胞和人类免疫缺陷病毒1型长时间重复（HIVLTR）中受到调节。一般策略是确定通过不同的阳性转录伸长因子B扮演的角色（p- Tefb）复合物，六甲基Blsacetimide诱导蛋白1和2（Hexim1/2），7SK小核 RNA（7SK RNA），核因子Kappa B（NF-KB），HIV转录反式激活器（TAT）和在此过程中，反式激活响应（TAR）RNA。 P-TEFB包含依赖细胞周期蛋白的激酶9 （CDK9）和C型细胞周期蛋白，CyCT1，Cyct2或Cyck。 NF-KB包含P65（RELA）和P50。特别重要性是包含的无效，高分子量（HMW）P-TEFB复合物之间的平衡另外，7SK RNA和Hexim1/2以及活跃的低分子量（LMW）P-TEFB复合物仅包含CDK9和C型细胞周期蛋白。具体的目的是尽可能精确地定义形成不活动的表面，HMW P-TEFB和活跃的LMW P-TEFB复合物结合NF-KB和TAT。 Hexim1/2的两个构象状态存在细胞，均与7SK RNA和P-TEFB复合，另一个是在溶液中不含的细胞。另外，我们想要确定Cyct1，Tat和Tar之间的复合物的精确三维结构，特别强调结合RNA和现在稳定的焦油的蛋白质表面。接下来，我们将研究这些不同P-TEFB复合物的功能以及如何通过翻译后调节它们修改。特别感兴趣的是Hexim1/2的磷酸化和泛素化。一旦激活， NF-KB如何招募P-TEFB到HIVLTR？ p65的泛素化在这方面起着什么作用过程？从其他研究中，我们知道单喹素结合Cyct1的C末端。这些研究将导致创建组成型活性hexim1/2蛋白质，该蛋白将用于确定其对细胞和病毒转录的差异影响。最后，我们将比较P-TEFB的招募 NF-KB通过DNA和TAT通过RNA在体外和体内，研究阴性之间的动态相互作用通过染色质免疫沉淀在HIVLTR上的伸长因子（N-TEF）和P-TEFB，以及在小鼠中编码Cyct1，Cyct2和Cyck的灭活基因。 P-TEFB的独特而多余的角色包含这些C型细胞周期蛋白的复合物将在生物体中揭示。这些研究将揭示P-TEFB的复杂RNA - 蛋白质世界，以及HIV如何颠覆这些细胞复合物在细胞中复制。