Figure 7
AtPUM-HD-2 demonstrates binding specificity to wildtype NRE1. EMSAs using purified GST-AtPum2 PUM-HD and wildtype or point mutated NRE1. (A) EMSA assays using the wildtype (NRE) or mutant (G to U) NRE1 RNA oligonucleotide in the absence or presence of GST or GST-AtPum2 PUM-HD (PUM). Unbound radiolabelled RNA (Free) shifts to a high molecular weight complex when bound to GST-AtPum2 PUM-HD (Bound). The fraction of bound RNA (Bf) was determined for each reaction. 100-fold excess non-labelled mutant NRE1 was added to the reaction containing labelled WT-NRE (NRE + comp). Conversely, 100-fold excess of non-labelled wildtype NRE1 was added to the reaction containing labelled mutant NRE RNA (G to U + comp). The underlined RNA sequence corresponds to cognate RNA that interacts with repeats 1 through 8 in the Drosophila Pumilio PUM-HD. (B) EMSA titration of WT-NRE1 and increasing concentrations of GST-AtPum2 PUM-HD (PUM-HD). The protein concentrations were 0, 0.12, 0.25, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125 and 250 nM. (C) The fraction of bound WT-RNA as a function of GST-AtPum2 PUM-HD from the EMSA in (B) was plotted and the dissociation constant (Kd) was determined. (D) EMSA titration of mutant NRE1 (G to U) and increasing concentrations of GST-AtPum2 PUM-HD. The protein concentrations were 0, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 nM.