Ping Y H, Rana T M
Department of Pharmacology, Robert Wood Johnson Medical School, and Molecular Biosciences Graduate Program at Rutgers University, Piscataway, New Jersey 08854, USA.
J Biol Chem. 2001 Apr 20;276(16):12951-8. doi: 10.1074/jbc.M006130200. Epub 2000 Dec 8.
Control of transcription elongation requires a complex interplay between the recently discovered positive transcription elongation factor b (P-TEFb) and negative transcription elongation factors, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) sensitivity inducing factors (DSIF) and the negative elongation factor (NELF). Activation of HIV-1 gene expression is regulated by a nascent RNA structure, termed TAR RNA, in concert with HIV-1 Tat protein and these positive and negative elongation factors. We have used a stepwise RNA pol II walking approach and Western blotting to determine the dynamics of interactions between HIV-1 Tat, DSIF/NELF, and the transcription complexes actively engaged in elongation. In addition, we developed an in vitro kinase assay to determine the phosphorylation status of proteins during elongation stages. Our results demonstrate that DSIF/NELF associates with RNA pol II complexes during early transcription elongation and travels with elongation complexes as the nascent RNA is synthesized. Our results also show that HIV-1 Tat protein stimulated DSIF and RNA pol II phosphorylation by P-TEFb during elongation. These findings reveal a molecular mechanism for the negative and positive regulation of transcriptional elongation at the HIV-1 promoter.
转录延伸的调控需要新发现的正性转录延伸因子b（P-TEFb）与负性转录延伸因子、5,6-二氯-1-β-D-呋喃核糖基苯并咪唑（DRB）敏感性诱导因子（DSIF）和负性延伸因子（NELF）之间复杂的相互作用。HIV-1基因表达的激活由一种新生RNA结构（称为TAR RNA）与HIV-1 Tat蛋白以及这些正性和负性延伸因子协同调控。我们采用逐步RNA聚合酶II步移方法和蛋白质印迹法来确定HIV-1 Tat、DSIF/NELF与积极参与延伸的转录复合物之间相互作用的动态变化。此外，我们开发了一种体外激酶测定法来确定延伸阶段蛋白质的磷酸化状态。我们的结果表明，DSIF/NELF在转录延伸早期与RNA聚合酶II复合物结合，并随着新生RNA的合成与延伸复合物一起移动。我们的结果还表明，HIV-1 Tat蛋白在延伸过程中刺激了P-TEFb介导的DSIF和RNA聚合酶II的磷酸化。这些发现揭示了HIV-1启动子处转录延伸的负性和正性调控的分子机制。
