Distinct DNA elements contribute to Rap1p affinity for its binding sites
Distinct DNA elements contribute to Rap1p affinity for its binding sites
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DOI：
10.1016/j.jmb.2004.03.047
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发表时间：
2004-05-14
影响因子：
5.6
通讯作者：
Negri, R
中科院分区：
文献类型：
--
作者：
Del Vescovo, V;De Sanctis, V;Negri, R
酿酒酵母（Saccharomycescerevisiae）的重要调控蛋白Rap1含有两个串联的Myb样DNA结合亚结构域，它们与两个确定的DNA“半位点”相互作用，并由一个三核苷酸接头序列隔开。我们已经映射了Rap1的DNA结合位点，结合结合和未结合的DNA的电泳分离的引物延伸方法。相对于已发表的共有序列，我们检测到超出推定DNA结合位点5 '端3个碱基对的结合相互作用。这个新的相互作用位点位于DNA小沟面对蛋白质的地方，并且可能解释了由Rap1p诱导的主要DNA弯曲，先前的研究已经映射到紧邻共识结合位点上游的位点。此外，我们表明，一个最小的DNA结合位点的一个单一的共识hemorrhage，之前或之后的间隔三核苷酸，与非结构化的蛋白质连接器之间的两个Rap1p的DNA结合结构域相互作用，是能够结合的蛋白质，虽然在较低的亲和力。这些发现可以解释观察到的Rap1p在许多启动子，缺乏典型的结合位点的体内结合特性。(C)2004爱思唯尔有限公司保留所有权利。
The essential Saccharomyces cerevisiae regulatory protein Rap1 contains two e tandem Myb-like DNA binding sub-domains that interact with two defined DNA "hemisites", separated by a trinucleotide linker sequence. We have mapped the thermodynamically defined DNA-binding site of Rap1 by a primer extension method coupled with electrophoretic separation of bound and unbound DNAs. Relative to published consensus sequences, we detect binding interactions that extend 3 bp beyond 5'-end of the putative DNA-binding site. This new site of interaction is located where the DNA minor groove faces the protein, and may account for the major DNA bending induced by Rap1p that previous studies have mapped to a site immediately upstream of the consensus binding site. In addition, we show that a minimal DNA-binding site made of one single consensus hemisite, preceded or followed by a spacer trinucleotide that interacts with the unstructured protein linker between the two Rap1p DNA binding domains, is able to bind the protein, although at lower affinity. These findings may explain the observed in vivo binding properties of Rap1p at many promoters that lack canonical binding sites. (C) 2004 Elsevier Ltd. All rights reserved.