Qiu Yijun, Gilmour David S
From the Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, Pennsylvania State University, University Park, Pennsylvania 16802.
From the Department of Biochemistry and Molecular Biology, Center for Eukaryotic Gene Regulation, Pennsylvania State University, University Park, Pennsylvania 16802
J Biol Chem. 2017 Mar 31;292(13):5555-5570. doi: 10.1074/jbc.M116.760751. Epub 2017 Feb 17.
DRB sensitivity-inducing factor (DSIF or Spt4/5) is a conserved transcription elongation factor that both inhibits and stimulates transcription elongation in metazoans. In and vertebrates, DSIF together with negative elongation factor (NELF) associates with RNA polymerase II during early elongation and causes RNA polymerase II to pause in the promoter-proximal region of genes. The mechanism of how DSIF establishes pausing is not known. We constructed Spt5 mutant forms of DSIF and tested their capacity to restore promoter-proximal pausing to DSIF-depleted nuclear extracts. The C-terminal repeat region of Spt5, which has been implicated in both inhibition and stimulation of elongation, is dispensable for promoter-proximal pausing. A region encompassing KOW4 and KOW5 of Spt5 is essential for pausing, and mutations in KOW5 specifically shift the location of the pause. RNA cross-linking analysis reveals that KOW5 directly contacts the nascent transcript, and deletion of KOW5 disrupts this interaction. Our results suggest that KOW5 is involved in promoter-proximal pausing through contact with the nascent RNA.
DRB敏感性诱导因子（DSIF或Spt4/5）是一种保守的转录延伸因子，在多细胞动物中既能抑制也能刺激转录延伸。在酵母和脊椎动物中，DSIF与负性延伸因子（NELF）在早期延伸过程中与RNA聚合酶II结合，并使RNA聚合酶II在基因的启动子近端区域暂停。DSIF如何建立暂停的机制尚不清楚。我们构建了DSIF的Spt5突变体形式，并测试了它们将启动子近端暂停恢复到DSIF缺失的细胞核提取物中的能力。Spt5的C末端重复区域，其在延伸的抑制和刺激中均有涉及，对于启动子近端暂停是可有可无的。包含Spt5的KOW4和KOW5的区域对于暂停至关重要，并且KOW5中的突变会特异性地改变暂停的位置。RNA交联分析表明KOW5直接与新生转录本接触，并且KOW5的缺失会破坏这种相互作用。我们的结果表明KOW5通过与新生RNA接触参与启动子近端暂停。
