Supplemental Information Molecular Cell, Volume 41

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1 Supplemental Information Molecular Cell, Volume 41 Molecular Mechanisms for the RNA-Dependent ATPase Activity of Upf1 and Its Regulation by Upf2 Sutapa Chakrabarti, Uma Jayachandran, Fabien Bonneau, Francesca Fiorini, Claire Basquin, Silvia Domcke, Hervé Le Hir, and Elena Conti

2 Figure S1. Sequence Conservation of Upf1 Structure-based sequence alignment of Upf1 from S. cerevisiae (S.c.), H. sapiens (H.s.), D. melanogaster (D.m.), S. pombe (S.p.) and the DEAD-box helicase Dbp5 from H. sapiens (H.s.). The secondary structural elements of S.c. Upf1 are represented as cylinders ( -helices) or arrows ( -strands) and colored according to the domains, as described in the text. Dashed lines represent disordered regions in the structure. Blue circles indicate the residues in the CH domain that bind to Upf2 (based on the structure of the Upf1-Upf2 complex, Clerici et al. 2009), black circles denote the residues that bind to RNA and the nucleotide binding residues are shown with black open triangles. The numbers 1, 2 and 3 denote coordination of zinc ions 1, 2 and 3. The helicase motifs are indicated. Residues conserved between Upf1 and other helicases like Dbp5 are shaded in dark violet while residues conserved across Upf1 orthologues are shaded in light violet.

3 Figure S2. Human Upf1 CH Is in an Open Conformation in the Absence of Nucleotide and RNA Structural superposition of the RecA1 domains of the human Upf1 CH in absence of RNA and nucleotide (yellow) and (A) phosphate-bound human Upf1 CH (grey, PDB coordinates 2GK7) (B) AMPPNP-bound human Upf1 CH (grey, PDB coordinates 2GJK) The RecA1 domains superpose with an r.m.s.d of 1 Å over 95% of all -carbon atoms. Note the conformational change of domain 1B and the relative orientation of the RecA. The inset of Figure S2B shows a stereo view of the details of the interaction of the RecA core with AMPPNP in the human Upf1 CH-AMPPNP complex. These interactions are identical to those mediated by the RecA core with ADP:AlF 4 in the transition-state complexes of Upf1 CH and Upf1 (see insets of Figures 2A and 2B).

4 Figure S3. Human and Yeast Upf1 Have Similar RNA Binding Properties (A) RNase protection assays of human and yeast Upf1 and Upf1 CH in presence of ADP:BeF 3. The Upf1 proteins of both species exhibit a similar behavior in that they protect more nucleotides of RNA than the corresponding Upf1 CH constructs (compare lane 1 to 3 and 2 to 4). The experiment was carried out as described previously in Bonneau et al., The asteriks (*) indicate the pattern generated by multiple molecules of Upf1 closely binding to the 60-mer RNA. (B) Native-PAGE analyses of complexes of human Upf1 with fluorescein tagged 30- and 15-mer poly(u) RNA. Protein and RNA were detected by Coomassie-staining and fluorescence scanning, respectively. The 15U RNA contains a single binding site for Upf1, while a longer RNA (30U) provides multiple Upf1-binding sites as indicated by the presence of higher order complexes. (C) Quantitative measurements of RNA-binding affinities of human and yeast Upf1 by fluorescence anisotropy. The experiments were performed in the absence of nucleotide and the data were fitted as described in the legend of Figure 1B. The Kds and their corresponding errors are the mean and standard deviation of five independent experiments. Human and yeast Upf1 exhibit similar low nanomolar affinities for RNA.

5 Figure S4. Integrity of the Critical Structural Features of Yeast Upf1-RNA-ADP:AlF 4 (A) Structural superposition of the CH domains of the yeast Upf1-RNA-ADP:AlF 4 complex (green) and the human Upf1-Upf2 complex (grey, PDB coordinates 2WJV). The CH domains superpose with an r.m.s.d of 1.3 Å over 90% of all -carbon atoms. The black spheres indicate the zinc ions in the yeast Upf1-RNA-ADP:AlF 4 complex and the numbers correlate them to the coordinating residues highlighted in Figure S1. (B) The inset of the top panel shows the F o F c omit electron density (contoured at 2.5 at the 3' end of the RNA. The inset of the bottom panel shows the 2F o F c electron density (contoured at 1 of the hydrophobic pocket made up of residues from the CH (green) and RecA2 (yellow) domains.