Volume 47, Issue 1, Pages 16-26 (July 2012) Structural Basis for Telomerase RNA Recognition and RNP Assembly by the Holoenzyme La Family Protein p65  Mahavir Singh, Zhonghua Wang, Bon-Kyung Koo, Anooj Patel, Duilio Cascio, Kathleen Collins, Juli Feigon  Molecular Cell  Volume 47, Issue 1, Pages 16-26 (July 2012) DOI: 10.1016/j.molcel.2012.05.018 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 47, 16-26DOI: (10.1016/j.molcel.2012.05.018) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Structure of p65 C-Terminal Domain (A) Domain structure of p65 and p65 C-terminal domain constructs. (B) Sequence and secondary structure of Tetrahymena TER and stem IV constructs. TBE indicates the template boundary element. (C–E) Solution NMR structure of p65-C2ΔL1: (C) ensemble of the 20 lowest energy NMR structures, (D) lowest energy structure; side view of β sheet. Atypical features are highlighted: α3 (red), β4′ (lime green), long β2-β3 loop (gray), nonaromatic residues at conserved positions on β3 RNP1 (cyan) and β1 RNP2 (violet), and location of the start of the C-terminal tail (red ★). (E) Cartoon rendering of lowest energy structure. The position of the β2-β3 loop is indicated by gray dots. (F) Crystal structure of p65-C1ΔL2 in cartoon rendering. In (C)–(F), the β sheet is orange, α3 is red, the β2-β3 loop is gray, and the rest of the protein is light orange. Molecular Cell 2012 47, 16-26DOI: (10.1016/j.molcel.2012.05.018) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Isothermal Calorimetry of p65 C-Terminal Domain with TER Stem IV (A–F) Isothermal calorimetry data and analysis for titration of S4 RNA into (A) p65-C1, (B) p65-C1ΔL1, (C) p65-C1ΔL2, (D) p65-C2, (E) p65-C1ΔL2 Y407A, and (F) p65-C1ΔL2 R465A. ITC data for other constructs are shown in Figure S5. Molecular Cell 2012 47, 16-26DOI: (10.1016/j.molcel.2012.05.018) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Crystal Structure of p65 C-Terminal Domain with TER Stem IV (A) Two views of the p65-C1ΔL2:S4 complex. (B) Schematic of the protein-RNA interactions. (C) Stick rendering of the RNA on the surface of the protein, illustrating the 105° bend induced by protein binding. (D) Protein interactions with the GA bulge. (E) View of helix α3x in the major groove. Aromatic side chains that wedge open the base pairs adjacent to the GA bulge and other residues that interact with the RNA are labeled. Protein color scheme is as in Figure 1. Gua, Ade, and Cyt that contact the protein are green, magenta, and cyan, respectively, and the rest of the RNA is light blue. Molecular Cell 2012 47, 16-26DOI: (10.1016/j.molcel.2012.05.018) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Comparison of Free and Bound Protein and RNA (A) Superposition of p65-C1ΔL2 free (green) and in complex with S4 (red). Side chains that form the GA bulge-binding pocket are shown. (B) The GA bulge residues in the protein-binding pocket. (C and D) Structures of the GA bulge and adjacent C-G base pairs in the (C) free RNA (PDB ID 2FEY) and (D) bound RNA, and schematics of the bend size and angle. RNA bend angles (tilt and roll) were measured using the program CURVES 5.1 (Lavery and Sklenar, 1988). Helix D and helix P represent the proximal and distal stem IV, respectively (see Figure 6C). Molecular Cell 2012 47, 16-26DOI: (10.1016/j.molcel.2012.05.018) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Hierarchical Assembly of p65, TERT, and TER (A–D) EMSA of TER with various p65-FL variants: (A) p65-FL, (B) p65-FLΔL1, (C), p65-FLΔL2, and (D) p65-ΔT. The bands corresponding to the migration of free TER and TER-p65 complexes are marked on right. At higher concentrations of p65-FL, p65-FLΔL1, and p65-FLΔL2, a second band corresponding to a supershifted p65-TER higher-order complex appears (O'Connor and Collins, 2006). (E–H) Stimulation of TERT binding to TER in the presence of p65-FL variants: (E) p65-FL, (F) p65-FLΔL1, (G) p65-FLΔL2, and (H) p65-FLΔT. For each assay, TERT-TER interaction was assayed on the same gel (lanes 1–5). TERT (1–516) concentration required to shift half of the p65-TER complexes is denoted using an asterisk. No stimulation of TERT interaction for TER was observed for p65-FLΔT-TER (H). The key identifying the various bands in (E)–(H) is shown at the right of (H). Molecular Cell 2012 47, 16-26DOI: (10.1016/j.molcel.2012.05.018) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 La and LARP7 Proteins Have a Common RRM2 Structure (A) Sequence alignment of the C-terminal region of three LARP7 family proteins, Tetrahymena telomerase p65 (Tt-p65), Euplotes telomerase p43 (Ea-p43), and human 7SK protein hLARP7 (Hs-LARP7); and human genuine La family protein, hLa (Hs-La). Secondary structure elements of p65 and hLa protein are shown above (orange and red) and below (blue) the sequence, respectively. The nonconsensus RNP1 and RNP2 are boxed in green with positions where conserved aromatic residues in typical RNPs would be marked with an asterisk. A conserved RNA binding motif on β2 identified in this study is labeled RNP3 and boxed in red. The red asterisks are conserved residues in RNP3, which in p65 xRRM2 interact with RNA. (B) Domain architecture of p65, p43, hLARP7, and hLa. (C) Model of the interaction of the LAM, RRM1, and xRRM2 domains of p65 on stem-loop IV. LAM and RRM1 domains are based on homology modeling with the crystal structure of hLa LAM and RRM1 complex with UUUA (PDB ID 2VOP), and loop IV is from PDB ID 2H2X. A schematic of the interactions is shown on the right. For description of the model building, see the Supplemental Experimental Procedures. Molecular Cell 2012 47, 16-26DOI: (10.1016/j.molcel.2012.05.018) Copyright © 2012 Elsevier Inc. Terms and Conditions