Hausner W, Thomm M
Institut für Allgemeine Mikrobiologie, Universität Kiel, Federal Republic of Germany.
J Biol Chem. 1993 Nov 15;268(32):24047-52.
We have recently shown that cell-free transcription of homologous templates from the archaeon Methanococcus thermolithotrophicus requires an archaeal transcription factor (aTFA) that separated from the RNA polymerase during phosphocellulose chromatography. We report here the identification and extensive purification of a second activity, aTFB, required for in vitro transcription. This activity copurified with RNA polymerase during initial chromatographic steps but was positively identified as a distinct transcription factor after Superdex 200 sizing chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the intensity of a M(r) = 28,000 polypeptide in silver-stained gels is correlated with transcription factor activity. The same polypeptide, when eluted from a denaturing polyacrylamide gel and subsequently renatured, showed the functional properties of the transcription factor. In conjunction with gel filtration and sedimentation studies, which indicated a molecular mass of 54,000 Da for the native protein, these results suggested that aTFB is a dimer with polypeptide chains of identical molecular mass. Functional studies with highly purified aTFB demonstrated that it is a general factor required for transcription of genes encoding tRNA and proteins.
我们最近发现，嗜热栖热甲烷球菌同源模板的无细胞转录需要一种古菌转录因子（aTFA），该因子在磷酸纤维素色谱过程中与RNA聚合酶分离。我们在此报告了体外转录所需的第二种活性物质aTFB的鉴定和大量纯化。这种活性在最初的色谱步骤中与RNA聚合酶共纯化，但在Superdex 200尺寸排阻色谱后被明确鉴定为一种独特的转录因子。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示，银染凝胶中分子量为28,000的多肽条带强度与转录因子活性相关。从变性聚丙烯酰胺凝胶上洗脱并随后复性的同一多肽，表现出转录因子的功能特性。结合凝胶过滤和沉降研究（结果表明天然蛋白的分子量为54,000 Da），这些结果表明aTFB是一种由分子量相同的多肽链组成的二聚体。对高度纯化的aTFB进行的功能研究表明，它是tRNA和蛋白质编码基因转录所需的通用因子。
