Henry N L, Sayre M H, Kornberg R D
Department of Cell Biology, Stanford University School of Medicine, California 94305.
J Biol Chem. 1992 Nov 15;267(32):23388-92.
Yeast RNA polymerase II general initiation factor g was purified to near homogeneity on the basis of its function in a reconstituted transcription system. Polypeptides of 30, 54, and 105 kDa co-purified with transcriptional activity, forming a complex with a mass of 300 kDa as judged by gel filtration, but only 100 kDa based on sedimentation in glycerol gradients, suggesting an elongated shape. Transcription activity could be reconstituted after separation of the three polypeptides under denaturing conditions; the 54- and 105-kDa subunits were both essential, while the 30-kDa subunit was slightly stimulatory. Factor g was required for initiation at all promoters tested, including those from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and adenovirus. Factor g can stably associate with RNA polymerase II, as shown by cosedimentation in a glycerol gradient.
酵母RNA聚合酶II通用起始因子g在重组转录系统中的功能基础上被纯化至近乎均一。30 kDa、54 kDa和105 kDa的多肽与转录活性共纯化，通过凝胶过滤判断形成了一个质量为300 kDa的复合物，但基于甘油梯度沉降仅为100 kDa，表明其形状呈细长状。在变性条件下分离这三种多肽后仍可重建转录活性；54 kDa和105 kDa的亚基都是必需的，而30 kDa的亚基有轻微的刺激作用。在所有测试的启动子上起始转录都需要因子g，包括来自酿酒酵母、粟酒裂殖酵母和腺病毒的启动子。如甘油梯度中共沉降所示，因子g可与RNA聚合酶II稳定结合。
