Yamaguchi Y, Wada T, Watanabe D, Takagi T, Hasegawa J, Handa H
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8501, Japan.
J Biol Chem. 1999 Mar 19;274(12):8085-92. doi: 10.1074/jbc.274.12.8085.
5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). We have previously identified and purified a novel transcription elongation factor, termed DSIF (for DRB sensitivity-inducing factor), that makes transcription sensitive to DRB. DSIF is composed of 160- and 14-kDa subunits, which are homologs of the Saccharomyces cerevisiae transcription factors Spt5 and Spt4. DSIF may either repress or stimulate transcription in vitro, depending on conditions, but its physiological function remains elusive. Here we characterize the structure and function of DSIF p160. p160 is shown to be a ubiquitous nuclear protein that forms a stable complex with p14 and interacts directly with the pol II largest subunit. Mutation analysis of p160 is used to identify structural features essential for its in vitro activity and to map the domains required for its interaction with p14 and pol II. Finally, a p160 mutant that represses DSIF activity in a dominant-negative manner is identified and used to demonstrate that DSIF represses transcription from various promoters in vivo.
5,6-二氯-1-β-D-呋喃核糖基苯并咪唑（DRB）是RNA聚合酶II（pol II）转录延伸的经典抑制剂。我们之前鉴定并纯化了一种新型转录延伸因子，称为DSIF（DRB敏感性诱导因子），它使转录对DRB敏感。DSIF由160 kDa和14 kDa亚基组成，它们是酿酒酵母转录因子Spt5和Spt4的同源物。DSIF在体外可能根据条件抑制或刺激转录，但其生理功能仍然不清楚。在这里，我们描述了DSIF p160的结构和功能。p160被证明是一种普遍存在的核蛋白，它与p14形成稳定的复合物，并直接与pol II最大亚基相互作用。对p160的突变分析用于确定其体外活性所必需的结构特征，并绘制其与p14和pol II相互作用所需的结构域。最后，鉴定出一种以显性负性方式抑制DSIF活性的p160突变体，并用于证明DSIF在体内抑制各种启动子的转录。
