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

项目总结/摘要 人7SK RNP是长的非编码7SK RNA和细胞蛋白的动态组装， 调节正转录延伸因子B（P-TEF B）的活性。P-TEFb是一种重要的真核生物 mRNA转录延伸的转录因子，其调节从启动子近端 暂停RNA聚合酶II（RNAPII）进入生产性延伸。P-TEFb也是一种必需的人类辅因子 用于HIV-1达特反式激活和病毒复制。人7SK核心RNP包含331个核苷酸， RNAPIII转录的非编码7SK RNA，一种称为MePCE的不寻常的甲基加帽酶， RNA 5'端的γ磷酸，以及与末端结合的La相关蛋白7，Larp 7， 发夹和UUU-3 'OH。在活性7SK snRNP、Hexim和P-TEFb中，细胞周期蛋白T1的异源二聚体和细胞周期蛋白T2的异源二聚体被抑制。 激酶Cdk 9结合7SK核心RNP;在该复合物中P-TEFb的相互作用通过螯合其活性而使其失活。 活性部位尽管7SK在mRNA、其他RNAPII RNA和HIV-1的转录调节中起着中心作用， 转录，相对较少的是知道在结构或机制的水平上如何细胞蛋白质组装 与7SK RNA形成功能性7SK RNP或达特如何与其相互作用以最终释放P-TEFb。我们将 采用核磁共振光谱学、X射线晶体学和低温电子显微镜的组合，沿着 生物化学方法研究7SK核心RNP（MePCE-7SK-Larp 7）的结构和组装 和7SK核心RNP加上Hexim和P-TEFb（“活动”7SK RNP），以便实现原子级 了解这一重要的宿主RNP对HIV-1病毒复制的作用。这些结构研究将奠定 为阐明7SK RNP背景下Tat-Hexim竞争的分子机制奠定了基础。 我们的目标是剖析潜在的中间步骤（即Tat结合的7SK RNP），导致P-TEFb劫持 从7SK RNP到HIV-1病毒超延伸复合物。这些实验的结果将提供 基本的分子见解和药物靶向的结构基础， 未表征的RNP是HIV-1转录所必需的，因此逃避潜伏期。

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.