Isolation and functional characterization of P-TEFb-associated factors that control general and HIV-1 transcriptional elongation.
Isolation and functional characterization of P-TEFb-associated factors that control general and HIV-1 transcriptional elongation.
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DOI：
10.1016/j.ymeth.2010.04.005
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发表时间：
2011-01
期刊：
影响因子：
--
通讯作者：
Zhou Q
中科院分区：
文献类型：
--
作者：
Chen R;Liu M;Zhang K;Zhou Q
最初被鉴定为RNA聚合酶（Pol）II转录延长细胞基因的关键因子，P-TEFb激酶随后被证明也是HIV-1转录所需的特异性宿主辅因子。通过溴结构域蛋白Brd 4募集到细胞启动子用于一般转录或HIV-1达特蛋白募集到病毒LTR用于活化HIV-1转录，P-TEFb通过磷酸化Pol II的C-末端结构域和一对负延伸因子刺激Pol II的持续合成能力，导致全长转录物的合成。然而，大量证据表明P-TEFb在细胞中并不单独起作用，其所有已知的生物学功能可能都是通过与各种调节剂的相互作用来介导的。虽然已经确定了一些P-TEFb相关因素，但可能还有更多尚未发现。鉴于P-TEFb在HIV-1转录中起着至关重要的作用，该领域面临的主要挑战是鉴定所有P-TEFb相关因子并确定它们如何调节Tat反式激活和HIV-1复制。这里描述的是一组实验程序，不仅使我们能够分离和鉴定几个P-TEFb相关因子，但也提供了手段来表征其在HIV-1转录控制的生化功能。鉴于最近的证据表明，转录延伸在控制后生动物基因表达中起着比以前认为的更重要的作用，这里提出的技术也将有助于分析Pol II延长细胞基因。
Originally identified as a factor crucial for RNA polymerase (Pol) II transcriptional elongation of cellular genes, the P-TEFb kinase was subsequently shown to also serve as a specific host co-factor required for HIV-1 transcription. Recruited by either the bromodomain protein Brd4 to cellular promoters for general transcription or the HIV-1 Tat protein to the viral LTR for activated HIV-1 transcription, P-TEFb stimulates the processivity of Pol II through phosphorylating the C-terminal domain of Pol II and a pair of negative elongation factors, leading to the synthesis of full-length transcripts. However, abundant evidence indicates that P-TEFb does not act alone in the cell and that all of its known biological functions are likely mediated through the interactions with various regulators. Although a number of P-TEFb-associated factors have already been identified, there are likely more yet to be discovered. Given that P-TEFb plays an essential role in HIV-1 transcription, a major challenge facing the field is to identify all the P-TEFb-associated factors and determine how they may modulate Tat-transactivation and HIV-1 replication. Described here is a set of experimental procedures that have not only enabled us to isolate and identify several P-TEFb-associated factors, but also provided the means to characterize their biochemical functions in HIV-1 transcriptional control. In light of the recent demonstrations that transcriptional elongation plays a much more important role in controlling metazoan gene expression than previously thought, the techniques presented here will also be useful for analyzing Pol II elongation of cellular genes.
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