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

Translational repression of hunchback (hb) mRNA in the posterior of the Dro sophila embryo requires two copies of a bipartite sequence, the Nanos Respo nse Element (NRE), located in the 3' untranslated region of the mRNA. The P UMILIO (PUM) protein is thought to bind the NREs and thereby repress hb tra nslation. 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-bind ing 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 t hat a dimer of PUM might recognize one NRE. Here, we analyze the RNA-bindin g affinity and stoichiometry of the DmPUM-HD and find that one DmPUM-HD mon omer binds independently and with equal affinity to each NRE (K-D similar t o 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 r ecognizes nucleotides in both the 5' and 3' NRE half-sites. Based on our es timate of the intraembryonic concentration of PUM (>40 nM), we propose that in vivo nearly all NREs are occupied by a PUM monomer.