Edwards A M, Darst S A, Feaver W J, Thompson N E, Burgess R R, Kornberg R D
Department of Cell Biology, Stanford School of Medicine, CA 94305.
Proc Natl Acad Sci U S A. 1990 Mar;87(6):2122-6. doi: 10.1073/pnas.87.6.2122.
Yeast RNA polymerase II was purified to homogeneity by a rapid procedure involving immunoaffinity chromatography. The purified enzyme contained 10 subunits, as reported for conventional preparations, but with no detectable proteolysis of the largest subunit. In assays of initiation of transcription at the yeast CYC1 promoter, the enzyme complemented the deficiency of an extract from a strain that produces a temperature-sensitive polymerase II. Mammalian RNA polymerase II was inactive in this initiation assay. The purified yeast enzyme formed two-dimensional crystals on positively charged lipid layers, as previously found for Escherichia coli RNA polymerase holoenzyme. Image analysis of electron micrographs of crystals in negative stain, which diffracted to about 30-A resolution, showed protein densities of dimensions consistent with those of single polymerase molecules.
通过一种涉及免疫亲和层析的快速方法，将酵母RNA聚合酶II纯化至同质状态。如常规制备方法所报道的那样，纯化后的酶含有10个亚基，但最大亚基未检测到蛋白水解现象。在酵母CYC1启动子处的转录起始测定中，该酶弥补了产生温度敏感型聚合酶II的菌株提取物的缺陷。哺乳动物RNA聚合酶II在此起始测定中无活性。纯化后的酵母酶在带正电荷的脂质层上形成二维晶体，这与之前在大肠杆菌RNA聚合酶全酶中发现的情况相同。对负染晶体的电子显微镜照片进行图像分析，其衍射分辨率约为30埃，结果显示蛋白质密度的尺寸与单个聚合酶分子的尺寸一致。
