Balciunas D, Gälman C, Ronne H, Björklund S
Department of Medical Biochemistry and Microbiology, Uppsala University Biomedical Center, Box 582, 751 23 Uppsala, Sweden.
Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):376-81. doi: 10.1073/pnas.96.2.376.
The mediator complex is essential for regulated transcription in vitro. In the yeast Saccharomyces cerevisiae, mediator comprises >15 subunits and interacts with the C-terminal domain of the largest subunit of RNA polymerase II, thus forming an RNA polymerase II holoenzyme. Here we describe the molecular cloning of the MED1 cDNA encoding the 70-kDa subunit of the mediator complex. Yeast cells lacking the MED1 gene are viable but show a complex phenotype including partial defects in both repression and induction of the GAL genes. Together with results on other mediator subunits, this implies that the mediator is involved in both transcriptional activation and repression. Similar to mutations in the SRB10 and SRB11 genes encoding cyclin C and the cyclin C-dependent kinase, a disruption of the MED1 gene can partially suppress loss of the Snf1 protein kinase. We further found that a lexA-Med1 fusion protein is a strong activator in srb11 cells, which suggests a functional link between Med1 and the Srb10/11 complex. Finally, we show that the Med2 protein is lost from the mediator on purification from Med1-deficient cells, indicating a physical interaction between Med1 and Med2.
中介体复合物对于体外的转录调控至关重要。在酿酒酵母中，中介体由超过15个亚基组成，并与RNA聚合酶II最大亚基的C末端结构域相互作用，从而形成RNA聚合酶II全酶。在此，我们描述了编码中介体复合物70-kDa亚基的MED1 cDNA的分子克隆。缺乏MED1基因的酵母细胞是有活力的，但表现出复杂的表型，包括GAL基因的阻遏和诱导方面的部分缺陷。结合关于其他中介体亚基的结果，这意味着中介体参与转录激活和阻遏。与编码细胞周期蛋白C和细胞周期蛋白C依赖性激酶的SRB10和SRB11基因中的突变类似，MED1基因的破坏可部分抑制Snf1蛋白激酶的缺失。我们进一步发现，lexA-Med1融合蛋白在srb11细胞中是一种强激活剂，这表明Med1与Srb10/11复合物之间存在功能联系。最后，我们表明，从缺乏Med1的细胞中纯化时，Med2蛋白会从中介体中丢失，这表明Med1与Med2之间存在物理相互作用。
