The PUMILIO-RNA interaction: A single RNA-Binding domain monomer recognizes a bipartite target sequence

被引:76
作者
Zamore, PD
Bartel, DP
Lehmann, R
Williamson, JR [1 ]
机构
[1] Scripps Res Inst, Skaggs Inst Chem Biol, La Jolla, CA 92037 USA
[2] MIT, Whitehead Inst Biomed Res, Cambridge, MA 02129 USA
[3] MIT, Dept Biol, Cambridge, MA 02129 USA
[4] NYU, Med Ctr, Skirball Inst, Howard Hughes Med Inst, New York, NY 10016 USA
关键词
D O I
10.1021/bi982264s
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (K-D similar to 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.
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页码:596 / 604
页数:9
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