Roles for RNA in Telomerase Nucleotide and Repeat Addition Processivity  Cary K. Lai, Michael C. Miller, Kathleen Collins  Molecular Cell  Volume 11, Issue 6, Pages 1673-1683 (June 2003) DOI: 10.1016/S1097-2765(03)00232-6

Figure 1 The 3′ Half of Tetrahymena Telomerase RNA Influences Nucleotide and Repeat Addition Processivity (A) Schematic of Tetrahymena telomerase RNA template and DNA products. The primer used here to enforce a unique template alignment, (TG)8TTGGG, is shown in capital letters; dNTPs added to synthesize product are shown in italic lower case letters. Product numbering refers to template positions during first repeat synthesis (+3 to +6) and to processive repeat addition in six-nucleotide increments (+12, etc.). (B) Schematic of the secondary structure of Tetrahymena thermophila telomerase RNA. In gray are the regions of telomerase RNA with known template-related functions. The positions of telomerase RNA 3′ truncations are indicated according to the residue number of the new 3′ end. Secondary structure elements are indicated in Roman numerals (stems I–IV; stem III forms a pseudoknot). (C) TERT was assembled with wild-type telomerase RNA (lane 1), 3′ truncated telomerase RNA (lanes 2–4), or no telomerase RNA (lane 5) before immunopurification and activity assay. Molecular Cell 2003 11, 1673-1683DOI: (10.1016/S1097-2765(03)00232-6)

Figure 2 Stem IV Stimulates Nucleotide and Repeat Addition Processivity (A) The structures of stems I and IV are shown. Nucleotides changed in the tetraloop substitution are shown in bold letters. (B) TERT was assembled with the stem IV truncation RNAs 1-107 (lanes 1–3) or 1-102 (lanes 4–6). Added to the activity assay was no additional RNA (lanes 1 and 4) or 108-159 RNA at 5 μM (lanes 2 and 5) or 20 μM (lanes 3 and 6). Repeat addition processivity (R.A.P.) is indicated relative to that of the wild-type enzyme. (C) Activity assays were performed using TERT assembled with wild-type telomerase RNA (lane 1), 3′ truncated telomerase RNA (lanes 2–6), or a UUCG tetraloop substitution of the distal loop of stem IV (lane 7). (D) TERT was assembled with 1-107 RNA and then assayed with no additional RNA (lane 1) or the indicated stem IV RNA at 2 μM (lanes 2, 4, and 6) or 10 μM (lanes 3, 5, and 7). Molecular Cell 2003 11, 1673-1683DOI: (10.1016/S1097-2765(03)00232-6)

Figure 3 The Stem III Pseudoknot Cooperates with Stem IV in Repeat Addition Processivity (A) Secondary structure schematics are shown for the telomerase RNA regions combined for complementation analysis. (B) TERT was assembled with 1-102 RNA (lanes 1–5) or 1-63 RNA (lanes 6–10). Subsequently, 0.5 μM or 2.5 μM of 103-159 RNA (lanes 2 and 3 and 7 and 8) or 64-159 RNA (lanes 4 and 5 and 9 and 10) was added to the activity assay. Repeat addition processivity (R.A.P.) is indicated relative to that of the wild-type enzyme. Molecular Cell 2003 11, 1673-1683DOI: (10.1016/S1097-2765(03)00232-6)

Figure 4 Pseudoknot Structure Is Important for Repeat Addition Processivity (A) Black lines and black dots indicate predicted pseudoknot base-pairing interactions. Bold letters indicate nucleotides that were modified in the pseudoknot variant RNAs. (B) Telomerase activity was assayed for RNPs assembled with wild-type telomerase RNA (lane 1), stem III deletions (lanes 2 and 3), sequence variants disrupting the base pairing of either pseudoknot stem (lanes 4 and 5 and 7 and 8), or complementary sequence variations restoring base-paired stems (lanes 6 and 9). Repeat addition processivity (R.A.P.) is indicated relative to that of the wild-type enzyme. The repeat addition processivity of RNP with CC77-78GG RNA could not be estimated due to the low level of activity. Molecular Cell 2003 11, 1673-1683DOI: (10.1016/S1097-2765(03)00232-6)

Figure 5 Low-Affinity TERT-Telomerase RNA Interactions at the TRE and Stem IV (A) The secondary structure of telomerase RNA with truncated RNA 3′ end positions and TERT interaction sites summarized. A schematic is also shown for cp107-42 RNA (a linker sequence connects the wild-type 5′ and 3′ RNA ends). (B) TERT protein was immunopurified after assembly with the indicated telomerase RNA variants; bound RNAs were recovered and analyzed by blot hybridization. TERT binding is quantitated relative to wild-type RNA. (C) Stem IV has a distributed TERT interaction site. TERT protein was immunopurified after assembly with circularly permuted telomerase RNAs that lacked the TRE and pseudoknot; bound RNAs were recovered and analyzed by blot hybridization. TERT binding is quantitated relative to cp107-42 RNA. Tetraloop indicates UUCG substitution of the indicated positions. Molecular Cell 2003 11, 1673-1683DOI: (10.1016/S1097-2765(03)00232-6)

Figure 6 Stem IV Contacts TERT Directly (A) Secondary structure of the cpRNA tested for TERT crosslinking. The ligated oligonucleotide region is shown in outline. (B) Activity assays were performed for RNPs assembled with cp143-142 RNA without 4-thiouracil substitution (lane 1) or with 4-thiouracil substitutions (lane 2). (C) Crosslinking was performed by UV irradiation at approximately 366 nm for 15 min and was followed by sample separation on a 10% SDS-PAGE gel. Migration of the RNA-TERT complex and RNA alone species are indicated by arrows. Molecular Cell 2003 11, 1673-1683DOI: (10.1016/S1097-2765(03)00232-6)

Figure 7 Proposed Roles for Telomerase RNA in the Telomerase Catalytic Cycle Molecular Cell 2003 11, 1673-1683DOI: (10.1016/S1097-2765(03)00232-6)