Ruet A, Camier S, Smagowicz W, Sentenac A, Fromageot P
EMBO J. 1984 Feb;3(2):343-50. doi: 10.1002/j.1460-2075.1984.tb01809.x.
A yeast extract was fractionated to resolve the factors involved in the transcription of yeast tRNA genes. An in vitro transcription system was reconstituted with two separate protein fractions and purified RNA polymerase C (III). Optimal conditions for tRNA synthesis have been determined. One essential component, termed tau factor, was partially purified by conventional chromatographic methods on heparin-agarose and DEAE-Sephadex; it sedimented as a large macromolecule in glycerol gradients (mol. wt. approximately 300 000). tau factor was found to form a stable complex with the tRNA gene in the absence of other transcriptional components. Complex formation is very fast, is not temperature dependent between 10 degrees C and 25 degrees C and does not require divalent cations. The factor-DNA complex is stable for at least 30 min at high salt concentration (0.1 M ammonium sulfate). These results indicate that gene recognition by a specific factor is a primary event in tRNA synthesis.
对酵母提取物进行分级分离，以解析参与酵母tRNA基因转录的因子。用两个单独的蛋白质组分和纯化的RNA聚合酶C（III）重建了体外转录系统。已确定了tRNA合成的最佳条件。一种称为tau因子的必需组分通过在肝素 - 琼脂糖和DEAE - 葡聚糖上的常规色谱方法进行了部分纯化；它在甘油梯度中作为大分子沉降（分子量约为300 000）。发现tau因子在没有其他转录组分的情况下与tRNA基因形成稳定的复合物。复合物的形成非常迅速，在10℃至25℃之间不依赖温度，并且不需要二价阳离子。在高盐浓度（0.1 M硫酸铵）下，因子 - DNA复合物至少稳定30分钟。这些结果表明，特定因子对基因的识别是tRNA合成中的首要事件。
