TRANSCRIPTIONAL INITIATION IS CONTROLLED BY UPSTREAM GC-BOX INTERACTIONS IN A TATAA-LESS PROMOTER
TRANSCRIPTIONAL INITIATION IS CONTROLLED BY UPSTREAM GC-BOX INTERACTIONS IN A TATAA-LESS PROMOTER
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DOI：
10.1128/mcb.10.12.6632
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发表时间：
1990-12-01
影响因子：
5.3
通讯作者：
AZIZKHAN, JC
中科院分区：
文献类型：
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作者：
BLAKE, MC;JAMBOU, RC;AZIZKHAN, JC
许多基因含有TATAA-less启动子，这类重要启动子对转录起始的控制尚不清楚。我们已经确定，在一个这样的启动子（仓鼠二氢叶酸还原酶基因）的四个近端GC盒序列元件中，有三个的蛋白质- dna相互作用控制了起始和主要和次要起始位点的相对使用。我们的结果表明，虽然GC盒在因子绑定方面显然是等效的，但它们在功能方面并不等效。转录起始需要至少两个位置正确的GC盒。通过最近的GC盒（盒I）的位点特异性突变消除dna -蛋白质相互作用，导致主要起始位点的转录减少了5倍，次要起始位点附近的异质转录增加了3倍，在体外和体内都是如此。在体外转录和瞬时表达试验中，分别取消GC盒II和III相互作用而保留GC盒I完整的突变影响了主要和次要起始位点的相对利用以及启动子模板的转录效率。当三个近端盒处于野生型配置时，GC盒IV的相互作用对二氢叶酸还原酶基因启动子的转录没有影响。因此，GC盒相互作用不仅是高效转录所必需的，而且还调节了这个TATAA-less启动子的起始位点利用。
Numerous genes contain TATAA-less promoters, and the control of transcriptional initiation in this important promoter class is not understood. We have determined that protein-DNA interactions at three of the four proximal GC box sequence elements in one such promoter, that of the hamster dihydrofolate reductase gene, control initiation and relative use of the major and minor start sites. Our results indicate that although the GC boxes are apparently equivalent with respect to factor binding, they are not equivalent with respect to function. At least two properly positioned GC boxes were required for initiation of transcription. Abolishment of DNA-protein interaction by site-specific mutation of the most proximal GC box (box I) resulted in a fivefold decrease in transcription from the major initiation site and a threefold increase in heterogeneous transcripts initiating from the vicinity of the minor start site in vitro and in vivo. Mutations that separately abolished interactions at GC boxes II and III while leaving GC box I intact affected the relative utilization of both the major and minor initiation sites as well as transcriptional efficiency of the promoter template in in vitro transcription and transient expression assays. Interaction of GC box IV when the three proximal boxes were in a wild-type configuration had no effect on transcription of the dihydrofolate reductase gene promoter. Thus, GC box interactions not only are required for efficient transcription but also regulate start site utilization in this TATAA-less promoter.