RRN3 gene of Saccharomyces cerevisiae encodes an essential RNA polymerase I transcription factor which interacts with the polymerase independently of DNA template.
RRN3 gene of Saccharomyces cerevisiae encodes an essential RNA polymerase I transcription factor which interacts with the polymerase independently of DNA template.
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DOI：
10.1002/j.1460-2075.1996.tb00770.x
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发表时间：
1996-08
期刊：
影响因子：
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通讯作者：
Robert TYamamoto;Yasuhisa Nogil;J. Dodd;Masayasu Nomura
中科院分区：
文献类型：
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作者：
Robert TYamamoto;Yasuhisa Nogil;J. Dodd;Masayasu Nomura
RRN 3是酿酒酵母中RNA聚合酶I（Pol I）转录rDNA所必需的RRN基因之一。我们已经克隆了该基因，确定了核苷酸序列，并发现它是一个必需的基因，编码的蛋白质的计算分子量为72 369。从rrn 3突变体制备的提取物在rDNA模板的体外转录中是有缺陷的。我们使用含有表位标记的Rrn 3蛋白的菌株的提取物来纯化可以补充突变体提取物的因子。使用免疫亲和纯化结合Mono Q色谱法，我们得到了一个基本上纯的制备Rrn 3 p的突变体提取物的补充。通过进行模板定型实验，我们发现Rrn 3 p不是通过多轮转录稳定的前起始复合物的一部分。我们还发现，Rrn 3 p与纯化的Pol I预孵育导致在随后与DNA模板和其他转录反应组分混合时刺激转录。使用洗涤剂Sarkosyl的单轮转录实验表明，这种刺激是由于Sarkosyl抗性预起始复合物形成的效率增加。因此，Rrn 3 p似乎直接与Pol I相互作用，显然刺激Pol I募集到启动子，并且与先前表征的另外两种Pol I特异性转录因子Rrn 6/7复合物和Rrn 5/9/10复合物（UAF）不同。
RRN3 is one of the RRN genes specifically required for the transcription of rDNA by RNA polymerase I (Pol I) in Saccharomyces cerevisiae. We have cloned the gene, determined the nucleotide sequence, and found that it is an essential gene which encodes a protein of calculated molecular weight of 72 369. Extracts prepared from rrn3 mutants were defective in in vitro transcription of rDNA templates. We used extracts from a strain containing an epitope‐tagged Rrn3 protein to purify a factor that could complement the mutant extracts. Using immunoaffinity purification combined with Mono Q chromatography, we obtained an essentially pure preparation of Rrn3p which complements the mutant extracts. By carrying out template commitment experiments, we found that Rrn3p is not part of the pre‐initiation complex that is stable through multiple rounds of transcription. We also found that pre‐incubation of Rrn3p with purified Pol I leads to stimulation of transcription upon subsequent mixing with DNA template and other transcription reaction components. Single‐round transcription experiments using the detergent Sarkosyl showed that this stimulation is due to increased efficiency of formation of a Sarkosyl‐resistant pre‐initiation complex. Thus, Rrn3p appears to interact directly with Pol I, apparently stimulating Pol I recruitment to the promoter, and is distinct from two other Pol I‐specific transcription factors, the Rrn6/7 complex and the Rrn5/9/10 complex (UAF), characterized previously.