The PUMILIO-RNA interaction: a single RNA-binding domain monomer recognizes a bipartite target sequence.
The PUMILIO-RNA interaction: a single RNA-binding domain monomer recognizes a bipartite target sequence.
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DOI：
10.1021/bi982264s
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发表时间：
1999-01
期刊：
影响因子：
2.9
通讯作者：
P. Zamore;D. Bartel;R. Lehmann;J. Williamson
中科院分区：
文献类型：
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作者：
P. Zamore;D. Bartel;R. Lehmann;J. Williamson
果蝇胚胎后部的驼背（hb）mRNA的翻译抑制需要两个双组分序列拷贝，即位于mRNA 3'非翻译区的纳米斯反应元件（NRE）。PUMILIO（PUM）蛋白被认为与NRE结合，从而抑制hb的翻译。PUM的RNA结合结构域定义了一个在进化上保守的RNA结合蛋白家族，即PUM同源结构域（PUM - HD）蛋白，它们已在酵母、植物和动物中被鉴定出来。PUM的RNA结合结构域，即果蝇PUM - HD（DmPUM - HD），先前已被证明能识别NRE的5'和3'部分的核苷酸，这表明PUM的二聚体可能识别一个NRE。在此，我们分析了DmPUM - HD的RNA结合亲和力和化学计量，发现一个DmPUM - HD单体独立地且以相等的亲和力与每个NRE结合（解离常数KD约为0.5 nM）。我们未检测到在相邻位点结合的DmPUM - HD单体之间存在协同相互作用。我们的结果意味着单个DmPUM - HD蛋白能识别5'和3' NRE半位点的核苷酸。根据我们对胚胎内PUM浓度（>40 nM）的估计，我们提出在体内几乎所有的NRE都被一个PUM单体占据。
Translational repression of hunchback (hb) mRNA in the posterior of the Drosophila embryo requires two copies of a bipartite sequence, the Nanos Response Element (NRE), located in the 3' untranslated region of the mRNA. The PUMILIO (PUM) protein is thought to bind the NREs and thereby repress hb translation. The RNA-binding domain of PUM defines an evolutionarily conserved family of RNA-binding proteins, the PUM-Homology Domain (PUM-HD) proteins, which have been identified in yeast, plants, and animals. The PUM RNA-binding domain, the Drosophila PUM-HD (DmPUM-HD), has been shown previously to recognize nucleotides in both the 5' and 3' halves of the NRE, suggesting that a dimer of PUM might recognize one NRE. Here, we analyze the RNA-binding affinity and stoichiometry of the DmPUM-HD and find that one DmPUM-HD monomer binds independently and with equal affinity to each NRE (KD approximately 0.5 nM). We detect no cooperative interactions between DmPUM-HD monomers bound at adjacent sites. Our results imply that a single DmPUM-HD protein recognizes nucleotides in both the 5' and 3' NRE half-sites. Based on our estimate of the intraembryonic concentration of PUM (>40 nM), we propose that in vivo nearly all NREs are occupied by a PUM monomer.