Zamore P D, Bartel D P, Lehmann R, Williamson J R
The Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge 02129, USA.
Biochemistry. 1999 Jan 12;38(2):596-604. doi: 10.1021/bi982264s.
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.
在果蝇胚胎后部，驼背（hb）mRNA的翻译抑制需要位于mRNA 3'非翻译区的两段二分序列，即纳米反应元件（NRE）。人们认为PUF蛋白（PUM）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，已在酵母、植物和动物中鉴定出来。PUM RNA结合结构域，即果蝇PUM-HD（DmPUM-HD），先前已被证明能识别NRE 5'和3'半段中的核苷酸，这表明PUM二聚体可能识别一个NRE。在这里，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单体独立地以相同亲和力结合每个NRE（KD约为0.5 nM）。我们未检测到相邻位点结合的DmPUM-HD单体之间的协同相互作用。我们的结果表明，单个DmPUM-HD蛋白识别NRE 5'和3'半位点中的核苷酸。基于我们对胚胎内PUM浓度（>40 nM）的估计，我们提出在体内几乎所有NRE都被一个PUM单体占据。
