SUMMARY Telomerase is a ribonucleoprotein complex that replicates the 3’ ends of linear chromosomes by successive additions of telomere repeat DNA. The telomerase holoenzyme contains two essential components for catalysis, a telomerase reverse transcriptase (TERT) and telomerase RNA (TER). The TER includes a template for telomere repeat synthesis as well as other domains required for function. We report the solution structure of the wild type minimal conserved human TER pseudoknot (PKWT) refined with an extensive set of RDCs, and a detailed analysis of the effect of the bulge U177 on pseudoknot structure, dynamics analyzed by RDC and 13C relaxation measurements, and base pair stability. The overall structure of PKWT is highly similar to the previously reported ΔU177 pseudoknot (PKDU) that has a deletion of a conserved bulge U important for catalytic activity. For direct comparison to PKWT, the structure of PKDU was re-refined with a comparable set of RDCs. Both pseudoknots contain a catalytically essential triple helix at the junction of the two stems, including two stem 1-loop 2 minor groove triples, a junction loop 1-loop 2 Hoogsteen base pair, and stem 2-loop 1 major groove U•A-U Watson-Crick-Hoogsteen triples located directly above the bulge U177. However there are significant differences in stabilities of base pairs near the bulge and the dynamics of some nucleotides. The stability of the base pairs in stem 2 surrounding the bulge U177 is greatly decreased, with the result that the Watson-Crick pairs in the triple helix begin to unfold before the Hoogsteen pairs, which may affect telomerase assembly and activity. The bulge U is positioned in the minor groove on the opposite face from the triple helical interactions, and sterically blocks the A176 2’OH which has recently been proposed to play a role in catalysis. The bulge U may serve as a hinge to provide backbone flexibility in this region.
Telomerase is a unique reverse transcriptase that catalyzes the addition of telomere DNA repeats onto the 3′ ends of linear chromosomes and plays a critical role in maintaining genome stability. Unlike other reverse transcriptases, telomerase is unique in that it is a ribonucleoprotein complex, where the RNA component [telomerase RNA (TR)] not only provides the template for the synthesis of telomere DNA repeats but also plays essential roles in catalysis, accumulation, TR 3′-end processing, localization, and holoenzyme assembly. Biochemical studies have identified TR elements essential for catalysis that share remarkably conserved secondary structures across different species as well as species-specific domains for other functions, paving the way for high-resolution structure determination of TRs. Over the past decade, structures of key elements from the core, conserved regions 4 and 5, and small Cajal body specific RNA domains of human TR have emerged, providing significant insights into the roles of these RNA elements in telomerase function. Structures of all helical elements of the core domain have been recently reported, providing the basis for a high-resolution model of the complete core domain. We review this progress to determine the overall architecture of human telomerase RNA.Box H/ACA RNA | NMR | pseudoknot | telomerase reverse transcriptase T elomerase is a large, multisubunit ribonucleoprotein (RNP) that replicates the 3′ end of linear chromosomes by processive synthesis of telomere DNA repeats. Telomeres, the physical ends of linear chromosomes, generally comprise dsDNA with a short repeating species-specific sequence ending in a 3′ single-stranded overhang of variable length plus associated telomere binding proteins, called shelterin in humans (1, 2). Telomeres protect the integrity of linear chromosomes by allowing the cellular DNA repair machinery to distinguish them from double-strand breaks, thus playing critical roles in maintaining genome stability in eukaryotes (1, 2). Shortening of telomeres below a critical length because of inherent incomplete replication of DNA ends ultimately leads to telomere fusions and cell senescence (3-6). The 3′ ends of telomeres are replicated by telomerase, a unique reverse transcriptase discovered almost three decades ago (7), which catalyzes the addition of telomere DNA repeats onto the ends of linear chromosomes using an embedded RNA as the template (8, 9). Although telomerase has a low or undetectable level of activity in most somatic cells, it is active in some germline, epithelial, and hematopoietic cells, and it is highly active in the majority (∼90%) of cancer cell lines (10-12). Telomerase deficiency because of mutations in human telomerase RNA (hTR) has also been linked to several inherited human diseases, such as dyskeratosis congenita, aplastic anemia, myelodysplasia, and idiopathic pulmonary fibrosis (13-26).The telomerase holoenzyme includes a unique reverse transcriptase [telomerase reverse transcriptase (TERT)], an essential RNA (TR), and several...
Telomerase is a unique ribonucleoprotein complex that catalyzes the addition of telomeric DNA repeats onto the 3′ ends of linear chromosomes. All vertebrate telomerase RNAs contain a catalytically essential core domain that includes the template and a pseudoknot with extended helical subdomains. Within these helical regions is an asymmetric 5-nt internal bulge loop (J2a/b) flanked by helices (P2a and P2b) that is highly conserved in its location but not sequence. NMR structure determination reveals that J2a/b forms a defined S-shape and creates an ∼90°bend with a surprisingly low twist (∼10°) between the flanking helices. A search of RNA structures revealed only one other example of a 5-nt bulge, from hepatitis C virus internal ribosome entry site, with a different sequence but the same structure. J2a/b is intrinsically flexible but the interhelical motions across the loop are remarkably restricted. Nucleotide substitutions in J2a/b that affect the bend angle, direction, and interhelical dynamics are correlated with telomerase activity. Based on the structures of P2ab (J2a/b and flanking helices), the conserved region of the pseudoknot (P2b/P3, previously determined) and the remaining helical segment (P2a.1-J2a.1 refined using residual dipolar couplings and the modeling program MC-Sym) we have calculated an NMR-based model of the fulllength pseudoknot. The model and dynamics analysis show that J2a/b serves as a dominant structural and dynamical element in defining the overall topology of the core domain, and suggest that interhelical motions in P2ab facilitate nucleotide addition along the template and template translocation.T elomeres are DNA-protein complexes that cap the ends of linear chromosomes. During each round of cell replication, telomeres shorten due to incomplete replication of telomere DNA repeats, and shortening of telomeres below a critical length leads to telomere fusions and cell senescence (1). Telomerase, a unique reverse transcriptase first discovered in Tetrahymena about two decades ago, is essential for maintaining the telomere length and the stability of chromosomes in most eukaryote species (2-4). A high level of telomerase activity is associated with cell proliferation in most (∼90%) cancers (5). The telomerase holoenzyme is a large complex comprising a unique reverse transcriptase protein (telomerase reverse transcriptase, TERT) which contains the active site for nucleotide addition, an essential RNA component [telomerase RNA (TR)] which contains the template for telomere DNA synthesis (6), and several speciesspecific proteins required for function in vivo such as assembly and localization (7).In addition to providing the template, the TR contains subdomains (8, 9) required for catalytic activity, localization, TR 3′ end processing, and accumulation (10). Telomerase deficiency due to mutations in TR has been linked to several inherited human diseases, such as dyskeratosis congenita, aplastic anemia, myelodysplasia, and idiopathic pulmonary fibrosis (11). The minimal components of verteb...
Telomerase is a unique reverse transcriptase that maintains the 3′ ends of eukaryotic chromosomes by adding tandem telomeric repeats. The RNA subunit (TR) of vertebrate telomerase provides a template for reverse transcription, contained within the conserved template/pseudoknot domain, and a conserved regions 4 and 5 (CR4/5) domain, all essential for catalytic activity. We report the nuclear magnetic resonance (NMR) solution structure of the full-length CR4/5 domain from the teleost fish medaka (Oryzias latipes). Three helices emanate from a structured internal loop, forming a Y-shaped structure, where helix P6 stacks on P5 and helix P6.1 points away from P6. The relative orientations of the three helices are Mg2+ dependent and dynamic. Although the three-way junction is structured and has unexpected base pairs, telomerase activity assays with nucleotide substitutions and deletions in CR4/5 indicate that none of these are essential for activity. The results suggest that the junction is likely to change conformation in complex with telomerase reverse transcriptase and that it provides a flexible scaffold that allows P6 and P6.1 to correctly fold and interact with telomerase reverse transcriptase.
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