Volume 19, Issue 9, Pages 1252-1261 (September 2011) Molecular Basis for the Recognition and Cleavage of RNA by the Bifunctional 5′–3′ Exo/Endoribonuclease RNase J  Audrey Dorléans, Inés Li de la Sierra-Gallay, Jérémie Piton, Léna Zig, Laetitia Gilet, Harald Putzer, Ciarán Condon  Structure  Volume 19, Issue 9, Pages 1252-1261 (September 2011) DOI: 10.1016/j.str.2011.06.018 Copyright © 2011 Elsevier Ltd Terms and Conditions

Structure 2011 19, 1252-1261DOI: (10.1016/j.str.2011.06.018) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 1 Structure of T. thermophilus RNase J Bound to RNA (A) Comparison of open and closed conformations. The enzyme is depicted as a monomer in ribbon conformation, with the β-lactamase domain in green, the β-CASP in violet, the linker domain in blue, and the C-terminal domain in pink. The 4 nt RNA is shown in stick conformation in yellow and the catalytic Zn ion in orange. The β-lactamase domain of the RNA-bound form has been superimposed on that of the free enzyme, in gray, to show the relative movement of the β-CASP and C-terminal domains (blue arrows). (B) Fo-Fc omit map for RNA at 2.5 σ above the mean. RNA nucleotides are in yellow, with phosphate groups in orange and red, and the Zn ion as an orange sphere. See also Figure S1. Structure 2011 19, 1252-1261DOI: (10.1016/j.str.2011.06.018) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 2 Movements within β-CASP and β-Lactamase Domains upon RNA Binding Stereo view of the movements in the loop between strand β15 and helix α9, the loop between strand β13 and helix α6, and the loop between strand β3 and strand β4. Secondary structure features of the β-CASP domain are shown in violet and those of the β-lactamase domain in green. RNA nucleotides are in yellow, with phosphate groups in orange and red. The equivalent features of the closed form are shown in gray. See also Figure S2. Structure 2011 19, 1252-1261DOI: (10.1016/j.str.2011.06.018) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 3 Schematic of Interactions between RNase J and RNA The nucleotides are labeled as −1, +1, +2, and +3 relative the position of cleavage. Amino acids making polar contacts (<3.6 Å) with the sugar-phosphate backbone are shown on the left, with the interacting groups specified in parentheses. The equivalent residues in B. subtilis RNase J1 and J2 are shown. Interactions with the bases are shown on the right, with the interacting group in parenthesis. See also Figure S3. Structure 2011 19, 1252-1261DOI: (10.1016/j.str.2011.06.018) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 4 5′ Phosphate-Binding Pocket of RNase J The 5′ phosphate-binding pocket is shown in the closed (A) and open (B) forms of RNase J. Residues are shown in stick form, with oxygens in red, nitrogen atoms in blue, and phosphates in orange. Residues from the β-lactamase domain are in green and those from the β-CASP domain in violet. A bridging water molecule is represented as a red sphere. Polar contacts are indicated by dashed lines. Structure 2011 19, 1252-1261DOI: (10.1016/j.str.2011.06.018) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 5 A Role of Key Residue Phe43 in Orienting Bases as They Move Toward the Catalytic Site The RNA is shown as yellow sticks and the side chain of Phe43 in blue. The β-lactamase and β-CASP domains are in green and violet ribbons, respectively; the Zn ion is represented as an orange sphere. Structure 2011 19, 1252-1261DOI: (10.1016/j.str.2011.06.018) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 6 RNA-Binding Channel and Proposed Nucleotide Exit Tunnel of RNase J (A) Representation of the three channels of RNase J as calculated by Caver. Channels are represented by hollow yellow tunnels, the β-lactamase domain in green ribbons, and the β-CASP domain in violet ribbons. The protein is shown in its monomeric form, and the C-terminal domain has been removed for clarity. The RNA is shown in red. (B) Slab view showing electrostatic surface predictions of RNase J, using the same view as in (A). Positively charged surfaces are shown in blue and negatively charged surfaces in red. The RNA is shown in yellow. The RNA-binding channel and proposed nucleotide exit tunnel are indicated. (C) Slab view showing 2 extra nt (orange) modeled in endonucleolytic-binding mode. (D) Surface view showing RNA bound to RNase J dimer. The β-lactamase (Bla) domain of each subunit is in green, the β-CASP domain in violet, the C terminus (Cter) in magenta, and the linker region in cyan. RNA is in red. The relative orientations to the overview shown in Figure 1A are indicated in the top left-hand corner for (A)–(D). See also Figure S6. Structure 2011 19, 1252-1261DOI: (10.1016/j.str.2011.06.018) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 7 Electrostatic Surface Model of B. subtilis RNase J1/J2 Heterodimer RNase J2 (right) is outlined in black. Positively charged surfaces are shown in blue and negatively charged surfaces in red. The proposed path to the catalytic cleft on the β-CASP is indicated. The 3′ end of the RNA (green) is labeled. RNA bound to RNase J2 is on the reverse side and is not visible. Structure 2011 19, 1252-1261DOI: (10.1016/j.str.2011.06.018) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 8 Kinetic Analysis of B. subtilis RNase J1/J2 Complex on 5′ and 3′-Labeled RNAs of Various Lengths (A) 5′-Labeled RNAs and (B) 3′-labeled RNAs. The lengths of the RNAs and the times of incubation are given above each autoradiogram. The band assignments were made by counting the visible bands between the mononucleotide product and the known number of nucleotides in the oligonucleotide substrate. Initial reaction rates are shown in the histogram to the right. The migration position of various mono-, di-, tri-, and tetra-nucleotides is shown. See also Figure S5. Structure 2011 19, 1252-1261DOI: (10.1016/j.str.2011.06.018) Copyright © 2011 Elsevier Ltd Terms and Conditions