Structural Basis of 3′ End RNA Recognition and Exoribonucleolytic Cleavage by an Exosome RNase PH Core  Esben Lorentzen, Elena Conti  Molecular Cell  Volume 20, Issue 3, Pages 473-481 (November 2005) DOI: 10.1016/j.molcel.2005.10.020 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 View of the S. solfataricus Rrp41-Rrp42 Exosome Core Bound to a Short Single-Stranded RNA (A) Overall view of the structure at the front side (where the active sites are located), with Rrp41 in blue and Rrp42 in green. The RNA is bound with the 3′ end near the phosphate binding site (where chloride is present, shown in magenta) and with the 5′ end toward the central channel. The protein is shown in ribbon representation on the left-hand side and in surface representation on right-hand side. This figure and all others representing structures were generated with PYMOL (http://pymol.sourceforge.net, Warren L. DeLano). (B) Detailed view of the structure at the RNA binding site, harboring subsites for four nucleotides (labeled N1–N4 from the 3′ end to the 5′ end). Amino acid residues crucial for RNA binding and catalysis are labeled. On the right, the molecule has been rotated 90° around the vertical axis. Molecular Cell 2005 20, 473-481DOI: (10.1016/j.molcel.2005.10.020) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 RNA Binding Arginines Are Essential for Catalytic Activity (A) Schematic of the interactions at the RNA binding site. Rrp41 residues are boxed in blue, Rrp42 in green, and the chloride ion in magenta. Black dotted lines represent electrostatic interactions between protein side chains and the RNA; gray dotted lines represent polar interactions engaging the main chain of the protein. (B) Degradation assay of wild-type Rrp41-Rrp42 and mutants on an A10 RNA substrate. The upper panel shows a denaturing 20% polyacrylamide gel where the nucleic acids have been visualized with toluidine. The lower panel is a Coomassie-stained gel showing the proteins incubated in the reaction mixture. (C) Conservation of important structural and functional residues in the RNase PH family. Structure-based sequence analysis identifies four regions (I–IV) of homology among S. solfataricus Rrp41 and Rrp42, human and yeast exosome proteins, and S. antibioticus PNPase and B. subtilis RNase PH. In orange are residues involved in structural interactions, either intramolecular or intermolecular. In red is a catalytic residue of Rrp41 (D182), and in pink are phosphate binding residues. In cyan are positively charged residues involved in binding the RNA phosphate backbone. Molecular Cell 2005 20, 473-481DOI: (10.1016/j.molcel.2005.10.020) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Reaction Mechanism (A) View of the substrate in the A5 exosome bound structure. The Fo–Fc simulated annealing electron density map is contoured at 2.5σ and shown with the final model superimposed. The orientation is similar to that of Figure 1B, left panel. (B) Structure of the cleavage product in the U8 exosome bound structure. The molecule is viewed in the same orientation as in (A) (see position of active site residues of Rrp41, D182, and the 134–138 loop). (C) Structure of the product ADP bound to the exosome in the same view as (A) and (B). (D) Schematic of the reaction mechanism. The Rrp41 residues are shown in blue, RNA in black, and the attacking inorganic phosphate in magenta. Molecular Cell 2005 20, 473-481DOI: (10.1016/j.molcel.2005.10.020) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Exosome Core Degradation Activity on RNA Substrates with a Stem-Loop Structure (A) Halved exosome core showing the central channel. The active site cavity is mostly shielded from solvent and accessible only via the central channel. The central channel is 25 Å wide at the front side but is constricted into four holes of 8–10 Å on the back side. (B) Exosome core degradation activity on substrates with a stem-loop structure. Degradation assay of wild-type Rrp41-Rrp42 with structured RNAs containing the same stem-loop structure (eight GC base pairs and a tetraloop) with different poly(A) extensions of 25 nucleotides (lanes 1 and 2), 15 nucleotides (lanes 3 and 4), 10 nucleotides (lanes 5 and 6), or 5 nucleotides (lanes 7 and 8). Molecular Cell 2005 20, 473-481DOI: (10.1016/j.molcel.2005.10.020) Copyright © 2005 Elsevier Inc. Terms and Conditions