Figure 2

Nucleotide binding and hydrolysis of hUpf1hd. (A) Comparison of the nucleotide-binding sites of hUpf1hd-AMPPNP and PcrA. hUpf1hd-AMPPNP and PcrA in the PcrA/ADPNP/dsDNA complex are colored in pink and orange, respectively. (B) ATPase activities of WT and mutant hUpf1hd proteins (200 nM) in the presence of 500 nM 15-mer poly(C). hUpf1hd showed very weak intrinsic ATPase activity without RNA. (C) ATP-binding activities of WT and mutant hUpf1hd proteins (15 g). (D) Northern analysis of the yeast CYH2 mRNA from strains expressing UPF1 mutant plasmids. mRNA from a UPF1 deletion strain containing plasmids expressing deletions (1B or 1C), empty vector (UPF1), WT, or point mutations as listed above each lane. The upper panel shows the unspliced precursor (upper band) and spliced CYH2 mRNA (lower band). The % of unspliced mRNA for each strain is given under each lane. The lower panel shows the 7S RNA used for standardization.