Human telomerase contains a 451 nt RNA (hTR) and several proteins, including a specialized reverse transcriptase (hTERT). The 5' half of hTR comprises the pseudoknot (core) domain, which includes the RNA template for telomere synthesis and a highly conserved pseudoknot that is required for telomerase activity. The solution structure of this essential pseudoknot, presented here, reveals an extended triple helix surrounding the helical junction. The network of tertiary interactions explains the phylogenetic sequence conservation and existing human and mouse TR functional studies as well as mutations linked to disease. Thermodynamic stability, dimerization potential, and telomerase activity of mutant RNAs that alter the tertiary contacts were investigated. Telomerase activity is strongly correlated with tertiary structure stability, whereas there is no correlation with dimerization potential of the pseudoknot. These studies reveal that a conserved pseudoknot tertiary structure is required for telomerase activity.

Autosomal dominant dyskeratosis congenita (DKC), as well as aplastic anemia, has been linked to mutations in the RNA component of telomerase, the ribonucleoprotein responsible for telomere maintenance. Here we examine the effect of the DKC mutations on the structure and stability of human telomerase RNA pseudoknot and CR7 domains by using NMR and thermal melting. The CR7 domain point mutation decreases stability and alters a conserved secondary structure thought to be involved in human telomerase RNA accumulation in vivo. We find that pseudoknot constructs containing the conserved elements of the pseudoknot domain are in equilibrium with a hairpin conformation. The solution structure of the wild-type hairpin reveals that it forms a continuous helix containing a novel run of three consecutive U⅐U and a U⅐C base pairs closed by a pentaloop. The six base pairs unique to the hairpin conformation are phylogenetically conserved in mammals, suggesting that this conformation is also functionally important. The DKC mutation in the pseudoknot domain results in a shift in the equilibrium toward the hairpin form, primarily due to destabilization of the pseudoknot. Our results provide insight into the effect of these mutations on telomerase structure and suggest that the catalytic cycle of telomerase involves a delicate interplay between RNA conformational states, alteration of which leads to the disease state. D yskeratosis congenita (DKC) is a rare inherited multisystemic disorder characterized by abnormal skin pigmentation, leukoplakia, and nail dystrophy (1). The known phenotypic hallmarks of DKC are defects in highly proliferative tissues that could ensue from a telomere maintenance disorder (2, 3). The leading causes of premature mortality due to DKC are progressive bone marrow failure, pulmonary disease, and malignancy. X-linked, autosomal recessive, and autosomal dominant inheritance patterns have been observed for DKC, and telomere lengths in patients with DKC are found to be significantly reduced in all three forms of inheritance (4). Similarly, some patients with ideopathic aplastic anemia also have shorter telomeres than age-matched controls (5). Although the X-linked form of DKC is characterized by mutations in the gene encoding the protein dyskerin (6), a component of the telomerase ribonucleoprotein (7), the autosomal dominant form of DKC has been linked to mutations in the gene encoding human telomerase RNA (hTR). The presence of telomerase RNA mutations and their segregation with the disease were confirmed in three families with DKC inheritance (8). One mutation results in deletion of the H͞ACA and CR7 domains (9) required for nucleolar localization, 3Ј-end processing, and RNA stability (7,(10)(11)(12), whereas the second is a point mutation (C408G) in the CR7 domain (Fig. 1a). The third mutation is a two-base substitution in the essential pseudoknot domain (9), which is required for activity (13,14) and is involved in the binding of the protein catalytic subunit (10) (Fig. 1a). Some patients with aplastic ...

Programmed -1 ribosomal frameshifting has become the subject of increasing interest over the last several years, due in part to the ubiquitous nature of this translational recoding mechanism in pathogenic animal and plant viruses. All cis-acting frameshift signals encoded in mRNAs are minimally composed of two functional elements: a heptanucleotide "slippery sequence" conforming to the general form X XXY YYZ, followed by an RNA structural element, usually an H-type RNA pseudoknot, positioned an optimal number of nucleotides (5 to 9) downstream. The slippery sequence itself promotes a low level ( approximately 1 %) of frameshifting; however, downstream pseudoknots stimulate this process significantly, in some cases up to 30 to 50 %. Although the precise molecular mechanism of stimulation of frameshifting remains poorly understood, significant advances have been made in our knowledge of the three-dimensional structures, thermodynamics of folding, and functional determinants of stimulatory RNA pseudoknots derived from the study of several well-characterized frameshift signals. These studies are summarized here and provide new insights into the structural requirements and mechanism of programmed -1 ribosomal frameshifting.

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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.