Structure of the Human Telomerase RNA Pseudoknot Reveals Conserved Tertiary Interactions Essential for Function  Carla A. Theimer, Craig A. Blois, Juli Feigon  Molecular Cell  Volume 17, Issue 5, Pages 671-682 (March 2005) DOI: 10.1016/j.molcel.2005.01.017 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 The Pseudoknot Domain of hTR RNA (A) 2° structure of hTR RNA with conserved subdomains identified (Chen et al., 2000). Nt conservation is indicated by green letters and bars (>95% for nt and bp, respectively), upper case black letters (>80%), and lower case letters (<80%), based on 51 sequences in the RFAM database (http://www.sanger.ac.uk/Software/Rfam/). Red letters and bars indicate the sites of disease-related mutations and deletions in the P2b-P3 region. (B) Sequence and 2° structure of the wt and ΔU177 RNA pseudoknot constructs. Cyan letters on the wt indicate nonnative sequences added during construct design. Coloring scheme of ΔU177 corresponds to Figure 2. (C) First derivative UV melting profiles of wt and ΔU177 RNAs with every second data point obtained at 260 and 280 nm shown as blue and red circles, respectively. The calculated fit to the data is indicated by solid (260 nm) and dashed (280 nm) black lines, with the 3° (indicated by arrow), stem 2, and stem 1 individual transitions shown in orange, green, and purple, respectively. Molecular Cell 2005 17, 671-682DOI: (10.1016/j.molcel.2005.01.017) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Solution Structure of the hTR Pseudoknot (A) Superposition of the 20 lowest energy structures and a ribbon representation of the backbone topology. (B) Stereoview of the lowest energy structure with the phosphate backbone identified by a gray ribbon. (C) Schematic representation of the pseudoknot junction and tertiary structure. (D) Stereoview of the junction region showing the network of stacked tertiary structural interactions. Orientation is the same as (C). Nt are colored by structural element: stem 1 (red), stem 2 (blue), loop 1 (orange), and loop 2 (green). Molecular Cell 2005 17, 671-682DOI: (10.1016/j.molcel.2005.01.017) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Individual Tertiary Structural Contacts in the hTR Pseudoknot (A) Two minor groove base triples, a Hoogsteen bp, and three Hoogsteen base triples are observed in the pseudoknot structure spanning the stem 1-stem 2 junction. Hydrogen bonds are indicated with dotted lines. (B) pH dependence of some pseudoknot mutations. pH points for the wt pseudoknot are in black, loop and stem mutations are colored by structural element as shown in Figure 2, and compensatory substitution data points are colored red with the fit to the data indicated by a solid black line. Molecular Cell 2005 17, 671-682DOI: (10.1016/j.molcel.2005.01.017) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Thermodynamic and Functional Consequences of Base Substitutions in the Pseudoknot Tertiary Structure (A) The telomerase activity of wt and mutant full-length-hTR-hTERT complexes were determined from replicate measurements by using the TRAP assay with bars indicating the average of three to five measurements normalized to wt activity and vertical error bars indicating the SD from repeated experiments. (B) The absolute value of the stability difference between wt and mutant pseudoknot sequences (|ΔΔG (37°C)| kcal mol−1). (C) The dimerization potential (dimer/[dimer + monomer]) of full-length wt and mutant pseudoknot sequences is the average of three measurements with vertical error bars indicating the SD from repeated experiments. (D and E) A fluorescence-based activity assay was used in which emission from a fluorescein-labeled primer indicates the level of amplified telomere product, and the assay is corrected by using (E) the emission from a sulforhodamine-labeled control primer and a PCR amplification control product. Emisson profiles are shown for wt (closed circles), ΔU177 (closed diamonds), and DKC (closed triangles) complexes as well as the telomerase positive (open down triangles), telomerase negative (plus signs), and no TAQ (open circles) controls. (F) Native gel showing dimerization of full-length wt, ΔU177, and DKC hTR RNAs. Samples were incubated on ice (0) or at 37°C (37) for 2 hr prior to loading. Monomer (M) and dimer (D) species are indicated to the right of the gel. Molecular Cell 2005 17, 671-682DOI: (10.1016/j.molcel.2005.01.017) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 A Conserved Tertiary Structure for the Pseudoknot (A) Schematic of the tertiary elements of a model of the mouse pseudoknot. (B) Solvent-accessible surface area of the pseudoknot structure. Nt are colored by structural element as in Figure 2. (C) Phylogenetic conservation. Nt that are >95% conserved are colored blue. (D and E) Chemical structure probing results mapped on the pseudoknot structure as oriented in (B) and with a 180° rotation in the plane of the paper. Surfaces colored pink and cyan indicate nt that are more or less reactive, respectively, to chemical modification in the free hTR versus telomerase complex. Surfaces colored yellow indicate uridines, which were not probed in the telomerase complex (Antal et al., 2002). Molecular Cell 2005 17, 671-682DOI: (10.1016/j.molcel.2005.01.017) Copyright © 2005 Elsevier Inc. Terms and Conditions