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

项目摘要/摘要 人类7SK RNP是非编码的长7SK RNA和细胞蛋白的动态组装， 调节正转录延伸因子b(P-TEFb)的活性。P-TEFb是一种必需的真核生物 调控启动子近端过渡的转录因子--mRNA转录延伸 暂停RNA聚合酶II(RNAPII)进入生产性伸长。P-TEFb也是一种重要的人类辅因子 用于HIV-1TAT的反式激活，从而实现病毒复制。人7SK核心RNP由331个核苷酸组成 RNAPIII-转录的非编码7SK RNA，一种名为MePCE的不寻常的甲基封顶酶，甲基化 RNA5‘末端的γ磷酸，以及与末端相关的La相关蛋白7，Larp7 发夹和UUU-3‘OH。在活性7SK SnRNP、Hexim和P-TEFb中，Cyclin T1和P-TEFb的杂二聚体 激酶CDK9，结合7SK核心RNP；P-TEFb在这个复合体中的相互作用通过隔离其 活动站点。尽管7SK在mRNA、其他RNAPII RNA和HIV-1的转录调控中起着中心作用 转录，在结构或机制水平上对细胞蛋白质如何组装知之甚少 与7SK RNA形成功能性7SK RNP，或Tat如何与其相互作用最终释放P-TEFb。我们会 使用核磁共振光谱、X射线结晶学和低温电子显微镜的组合，以及 生化方法研究7SK核心RNP(MePCE-7SK-Larp7)的结构和组装 以及7SK核心RNP加上Hexim和P-TEFb(主动的7SK RNP)，以实现原子级 了解这一重要宿主RNP对HIV-1病毒复制的影响。这些结构研究将为 为阐明7SK RNP背景下TAT-Hexim竞争的分子机制奠定了基础。 我们的目标是剖析导致P-TEFb劫持的潜在中间步骤(即TAT结合的7SK RNP) 从7SK RNP进入HIV-1病毒超延长复合体。这些实验的结果将提供 主要从结构上对药物靶向的基本分子洞察和结构基础 未知的RNP是HIV-1转录所必需的，因此可以逃避潜伏期。

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.