Paule M R, Iida C T, Perna P J, Harris G H, Brown Shimer S L, Kownin P
Biochemistry. 1984 Aug 28;23(18):4167-72. doi: 10.1021/bi00313a025.
A faithful transcription system for ribosomal RNA genes has been developed by using components from the small free-living amoeba Acanthamoeba castellanii. The system utilizes protein-free recombinant DNA as a template and in addition requires a crude cell-free extract containing RNA polymerase I and a transcription initiation factor (TIF-I). The transcript is initiated at the same position as the in vivo precursor ribosomal RNA: templates truncated at various sites downstream of the transcription start site give rise to only the predicted runoff RNA transcripts, and the runoff transcript produced has a 5'-terminus identical with the 5'-terminus of the isolated ribosomal RNA precursor. Faithful initiation can be elicited by the DNA sequence extending from -55 to +19 in the template. Subclones containing this sequence yield only the predicted runoff RNAs regardless of the orientation of this fragment in the cloning vector DNA; thus, only the in vivo sense strand of the template is specifically transcribed in the in vitro system. The system is specific for the RNA polymerase responsible for the transcription of ribosomal RNA genes in vivo. Faithful transcription, like RNA polymerase I from Acanthamoeba, is insensitive to alpha-amanitin inhibition, and transcription is greatly stimulated by highly purified RNA polymerase I but not by RNA polymerases II or III. Conditions for optimal transcription were determined.
通过使用来自小型自由生活变形虫卡氏棘阿米巴的成分，已开发出一种用于核糖体RNA基因的忠实转录系统。该系统以无蛋白质的重组DNA作为模板，此外还需要一种含有RNA聚合酶I和转录起始因子（TIF-I）的粗制无细胞提取物。转录本在与体内前体核糖体RNA相同的位置起始：在转录起始位点下游不同位点截短的模板仅产生预测的径流RNA转录本，并且产生的径流转录本的5'末端与分离的核糖体RNA前体的5'末端相同。从模板中-55至+19延伸的DNA序列可引发忠实起始。包含该序列的亚克隆无论该片段在克隆载体DNA中的方向如何，都仅产生预测的径流RNA；因此，在体外系统中仅特异性转录模板的体内有义链。该系统对负责体内核糖体RNA基因转录的RNA聚合酶具有特异性。与来自棘阿米巴的RNA聚合酶I一样，忠实转录对α-鹅膏蕈碱抑制不敏感，并且高度纯化的RNA聚合酶I可极大地刺激转录，而RNA聚合酶II或III则不能。确定了最佳转录条件。
