The yeast RNA polymerase I promoter: ribosomal DNA sequences involved in transcription initiation and complex formation in vitro.
The yeast RNA polymerase I promoter: ribosomal DNA sequences involved in transcription initiation and complex formation in vitro.
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酵母 RNA 聚合酶 I 启动子：参与体外转录起始和复合物形成的核糖体 DNA 序列。
DOI：
10.1093/nar/19.19.5363
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发表时间：
1991
影响因子：
14.9
通讯作者：
Nomura,M
中科院分区：
文献类型：
--
作者：
Kulkens,T;Riggs,DL;Heck,JD;Planta,RJ;Nomura,M
利用酵母RNA聚合酶I的体外转录系统，我们分析了Pol I启动子缺失突变体，并绘制了启动子-155和+27之间的边界。然而，能够转录起始的最小核心启动子的5 '边界被发现位于-38和-26之间。延伸至-2和-5的3′-缺失仍然允许一些转录，这表明Pol I的定位是由上游序列指导的。对连接子扫描突变体（linker scanning mutants，LSMs）的体外分析结合缺失分析的结果表明，该启动子由三个结构域组成：两个基本的核心结构域（I：− 2 8至+8和II：−76至−51）和一个转录调节上游结构域（III：−146至−91）。这些结果与体内实验结果基本一致（1）。使用模板竞争试验，我们还分析了这些突变启动子形成稳定的preinitiation复合物的能力。我们发现，5′-缺失突变体的能力，以螯合一个必要的因素（S）与他们的转录活性。相反，结构域I和II中的一些3′-缺失和一些LSM极大地降低转录活性，而不显著降低竞争能力。结果表明，结构域III的刺激功能是通过其与一个必需的转录因子的相互作用，虽然其他结构域也参与这种相互作用，可能直接或通过另一种蛋白质因子。
Using anin vitrotranscription system forSaccharomyces cerevisiaeRNA polymerase I, we have analyzed Pol I promoter deletion mutants and mapped the boundaries of the promoter between positions −155 and +27. The 5′-boundary of the minimal core promoter capable of transcription initiation, however, was found to lie between −38 and −26. The 3′-deletion extending to − 2 and − 5 still allowed some transcription, suggesting that the positioning of Pol I is directed by upstream sequences. The results ofin vitroanalysis of linker scanning mutants (LSMs) combined with the deletion analysis showed that the promoter consists of three domains: two essential core domains (I: − 2 8 to +8 and II: −76 to −51) and a transcription modulating upstream domain (III: −146 to −91). These results are in general agreement with those obtainedin vivo(1). Using a template competition assay we also analyzed these mutant promoters for their ability to form a stable preinitiation complex. We found that the ability of 5′-deletion mutants to sequester an essential factor(s) correlates with their transcriptional activity. In contrast, several 3′-deletions and some LSMs in domain I and II decrease transcription activity greatly without significantly decreasing competition ability. The results indicate that the stimulatory function of domain III is achieved through its interaction with an essential transcription factor(s), although the other domains also participate in this interaction, perhaps directly or through another protein factor.