Structural biology of 7SK RNP and its interaction with HIV-1 Tat

7SK RNP 的结构生物学及其与 HIV-1 Tat 的相互作用

基本信息

批准号：
10402809
负责人：
JULI FEIGON
金额：
$ 47.94万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10402809
关键词：
3-Dimensional Active Sites Binding Binding Proteins Binding Sites Biochemical C-terminal CDK9 Protein Kinase Cell Maintenance Complement Factor B Complex Cryoelectron Microscopy Cyclin-Dependent Kinases Disease Drug Targeting Elongation Factor Enzymes Genetic Transcription Goals HIV HIV-1 Health Heart Hypertrophy Human In Vitro Laboratories Lead Link Messenger RNA Methods Methylation Molecular Molecular Conformation NMR Spectroscopy Phosphotransferases Play Positive Transcriptional Elongation Factor B Proteins RNA RNA Binding RNA Conformation RNA Polymerase II RNA Recognition Motif RNA-Protein Interaction Regulation Reporting Resistance Role Small Nuclear Ribonucleoproteins Stress Structure Trans-Activators Transactivation Transcription Elongation Transcription Initiation Transcriptional Regulation U6 small nuclear RNA Untranslated RNA Viral Virus Replication Work X-Ray Crystallography circular RNA cofactor cyclin T1 experimental study in vivo inorganic phosphate insight leukemia/lymphoma promoter protein complex recruit stoichiometry structural biology transcription factor

项目摘要

PROJECT SUMMARY/ABSTRACT The human 7SK RNP is a dynamic assembly of the long non-coding 7SK RNA and cellular proteins that regulates the activity of positive transcription elongation factor b (P-TEFb). P-TEFb is an essential eukaryotic transcription factor for mRNA transcription elongation, which regulates the transition from promoter proximal paused RNA polymerase II (RNAPII) into productive elongation. P-TEFb is also an essential human cofactor for HIV-1 Tat transactivation and therefore viral replication. The human 7SK core RNP comprises the 331 nt RNAPIII-transcribed non-coding 7SK RNA, an unusual methyl capping enzyme called MePCE that methylates the γ phosphate on the RNA 5' terminus, and the La related protein 7, Larp7, that associates with the terminal hairpin and UUU-3'OH. In the active 7SK snRNP, Hexim and P-TEFb, a heterodimer of Cyclin T1 and the kinase Cdk9, bind the 7SK core RNP; interaction of P-TEFb in this complex inactivates it by sequestering its active site. Despite the central role of 7SK in transcription regulation of mRNA, other RNAPII RNAs, and HIV-1 transcription, relatively little is known at a structural or mechanistic level about how cellular proteins assemble with 7SK RNA to form a functional 7SK RNP or how Tat interacts with it to ultimately release P-TEFb. We will employ a combination of NMR spectroscopy, X-ray crystallography, and cryo electron microscopy along with biochemical methods to investigate the structures and assembly of the 7SK core RNP (MePCE–7SK–Larp7) and 7SK core RNP plus Hexim and P-TEFb (the `active' 7SK RNP) in order to achieve an atomic-level understanding of this important host RNP for HIV-1 viral replication. These structural studies will lay the groundwork for elucidating the molecular mechanisms of Tat-Hexim competition in the context of 7SK RNP. We aim to dissect the potential intermediate steps (i.e. Tat-bound 7SK RNP) that lead to P-TEFb hijacking from 7SK RNP into the HIV-1 viral super-elongation complex. The results of these experiments will provide fundamental molecular insights into and a structural basis for drug targeting of this largely structurally uncharacterized RNP that is essential for HIV-1 transcription and therefore escape from latency.

项目摘要/摘要人7sk RNP是长期非编码7SK RNA和细胞蛋白的动态组装调节阳性转录伸长因子B（P-TEFB）的活性。 P-TEFB是必不可少的真核生物 mRNA转录伸长的转录因子，调节启动子代理的过渡暂停的RNA聚合酶II（RNAPII）成生产性延伸。 P-TEFB也是必不可少的人类辅助因子用于HIV-1 TAT反式激活，因此病毒复制。人7SK核心RNP包括331 nt rnapiii transcranced非编码7SK RNA，一种称为MEPCE的不寻常的甲基上限酶，该酶是甲基化的 RNA 5'末端上的γ磷酸盐和与末端相关的LA相关蛋白7，LARP7 发夹和uuu-3'oh。在活跃的7SK SNRNP中，Hexim和P-TEFB，Cyclin T1的异二聚体激酶CDK9，结合7SK Core RNP； P-TEFB在该复合物中的相互作用通过隔离它活性站点。尽管7SK在mRNA，其他RNAPII RNA和HIV-1中的转录调节中具有中心作用转录在结构或机械水平上相对较少，以了解细胞蛋白如何组装使用7SK RNA形成功能性7SK RNP或TAT如何与之相互作用以最终释放P-TEFB。我们将员工NMR光谱，X射线晶体学和冷冻电子显微镜的组合以及研究7SK Core RNP的结构和组装的生化方法（MEPCE – 7SK – LARP7）以及7SK Core RNP加Hexim和P-TEFB（“ Active” 7SK RNP），以实现原子级了解HIV-1病毒复制的这一重要宿主RNP。这些结构研究将奠定在7SK RNP的背景下阐明Tat-Hexim竞争的分子机制的基础。我们旨在剖析导致P-TEFB劫持的潜在中间步骤（即tat-bound 7SK RNP）从7SK RNP到HIV-1病毒超延长复合物。这些实验的结果将提供基本的分子见解和靶向该药物的结构性基础在很大程度上在结构上对HIV-1转录至关重要的未表征的RNP，因此逃脱了潜伏期。