Thermodynamic characterization of the Saccharomyces cerevisiae telomerase RNA pseudoknot domain in vitro

Recent structural and functional characterization of the pseudoknot in the Saccharomyces cerevisiae telomerase RNA (TLC1) has demonstrated that tertiary structure is present, similar to that previously described for the human and Kluyveromyces lactis telomerase RNAs. In order to biophysically characterize the identified pseudoknot secondary and tertiary structures, UV-monitored thermal denaturation experiments, nuclear magnetic resonance spectroscopy, and native gel electrophoresis were used to investigate various potential conformations in the pseudoknot domain in vitro, in the absence of the telomerase protein. Here, we demonstrate that alternative secondary structures are not mutually exclusive in the S. cerevisiae telomerase RNA, tertiary structure contributes 1.5 kcal mol(-1) to the stability of the pseudoknot (≈ half the stability observed for the human telomerase pseudoknot), and identify additional base pairs in the 3' pseudoknot stem near the helical junction. In addition, sequence conservation in an adjacent overlapping hairpin appears to prevent dimerization and alternative conformations in the context of the entire pseudoknot-containing region. Thus, this work provides a detailed in vitro characterization of the thermodynamic features of the S. cerevisiae TLC1 pseudoknot region for comparison with other telomerase RNA pseudoknots.     

A human-Tetrahymena pseudoknot chimeric telomerase RNA reconstitutes a nonprocessive enzyme in vitro that is defective in telomere elongation

The phylogenetically-derived secondary structures of telomerase RNAs (TR) from ciliates, yeasts and vertebrates are surprisingly conserved and contain a pseudoknot domain at a similar location downstream of the template. As the pseudoknot domains of Tetrahymena TR (tTR) and human TR (hTR) mediate certain similar functions, we hypothesized that they might be functionally interchangeable. We constructed a chimeric TR (htTR) by exchanging the hTR pseudoknot sequences for the tTR pseudoknot region. The chimeric RNA reconstituted human telomerase activity when coexpressed with hTERT in vitro, but exhibited defects in repeat addition processivity and levels of DNA synthesis compared to hTR. Activity was dependent on tTR sequences within the chimeric RNA. htTR interacted with hTERT in vitro and dimerized predominantly via a region of its hTR backbone, the J7b/8a loop. Introduction of htTR in telomerase-negative cells stably expressing hTERT did not reconstitute an active enzyme able to elongate telomeres. Thus, our results indicate that the chimeric RNA reconstituted a weakly active nonprocessive human telomerase enzyme in vitro that was defective in telomere elongation in vivo. This suggests that there may be species-specific requirements for pseudoknot functions.     

Human telomerase RNA pseudoknot and hairpin thermal stability with glycine betaine and urea: preferential interactions with RNA secondary and tertiary structures

Thermal denaturation of the human telomerase RNA (hTR) DeltaU177 pseudoknot and hTR p2b hairpin was investigated by dual UV-wavelength absorbance spectroscopy in aqueous glycine betaine and urea solutions. The hTR DeltaU177 pseudoknot contains two helix-loop interactions that comprise the tertiary structure, as well as a GC-rich 6 bp stem (stem 1) and an AU-rich 9 bp stem (stem 2). The p2b hairpin also contains GC-rich stem 1 and a unique uridine-rich helix with a pentaloop. Glycine betaine stabilizes the pseudoknot tertiary structure in 135 mm NaCl and facilitates only a minor destabilization of tertiary structure in 40 mm NaCl. As with double-helical DNA, glycine betaine interacts more strongly with the surface area exposed upon unfolding of GC-rich stem 1 than either AU-rich stem 2 or the hairpin uridine-rich helix. Urea was shown to destabilize all RNA pseudoknot and hairpin secondary and tertiary structures but exhibits a stronger preferential interaction with AU-rich stem 2. Correlating these interactions with water-accessible surface area calculations indicates that the extent of interaction of glycine betaine with the surface area exposed upon RNA unfolding decreases as the nonpolar character of the unfolded RNA surface increases. As expected, the extent of interaction of urea with the surface area exposed for unfolding RNA increases as the fraction of amide functional groups increases. However, interaction of urea with amide functional groups alone cannot explain the stronger preferential interaction of urea with AU-rich stem 2. Interaction of urea with adenine relative to guanine and cytosine bases or sequence-dependent hydration is proposed for the stronger preferential interaction of urea with AU-rich duplexes.     

Biphasic folding kinetics of RNA pseudoknots and telomerase RNA activity

Using a combined master equation and kinetic cluster approach, we investigate RNA pseudoknot folding and unfolding kinetics. The energetic parameters are computed from a recently developed Vfold model for RNA secondary structure and pseudoknot folding thermodynamics. The folding kinetics theory is based on the complete conformational ensemble, including all the native-like and non-native states. The predicted folding and unfolding pathways, activation barriers, Arrhenius plots, and rate-limiting steps lead to several findings. First, for the PK5 pseudoknot, a misfolded 5' hairpin emerges as a stable kinetic trap in the folding process, and the detrapping from this misfolded state is the rate-limiting step for the overall folding process. The calculated rate constant and activation barrier agree well with the experimental data. Second, as an application of the model, we investigate the kinetic folding pathways for human telomerase RNA (hTR) pseudoknot. The predicted folding and unfolding pathways not only support the proposed role of conformational switch between hairpin and pseudoknot in hTR activity, but also reveal molecular mechanism for the conformational switch. Furthermore, for an experimentally studied hTR mutation, whose hairpin intermediate is destabilized, the model predicts a long-lived transient hairpin structure, and the switch between the transient hairpin intermediate and the native pseudoknot may be responsible for the observed hTR activity. Such finding would help resolve the apparent contradiction between the observed hTR activity and the absence of a stable hairpin.     

Mutational analysis of the Tetrahymena telomerase RNA: identification of residues affecting telomerase activity in vitro.

Telomere-specific repeat sequences are essential for chromosome end stability. Telomerase maintains telomere length by adding sequences de novo onto chromosome ends. The template domain of the telomerase RNA component dictates synthesis of species-specific telomeric repeats and other regions of the RNA have been suggested to be important for enzyme structure and/or catalysis. Using enzyme reconstituted in vitro with RNAs containing deletions or substitutions we identified nucleotides in the RNA component that are important for telomerase activity. Although many changes to conserved features in the RNA secondary structure did not abolish enzyme activity, levels of activity were often greatly reduced, suggesting that regions other than the template play a role in telomerase function. The template boundary was only altered by changes in stem II that affected the conserved region upstream of the template, not by changes in other regions, such as stems I, III and IV, consistent with a role of the conserved region in defining the 5' boundary of the template. Surprisingly, telomerase RNAs with substitutions or deletion of residues potentially abolishing the conserved pseudoknot structure had wild-type levels of telomerase activity. This suggests that this base pairing interaction may not be required for telomerase activity per se but may be conserved as a regulatory site for the enzyme in vivo.     

Telomerase Activity Is Sensitive to Subtle Perturbations of the TLC1 Pseudoknot 3′ Stem and Tertiary Structure

Pseudoknot formation in the core region of the telomerase RNA has been demonstrated to be important for telomerase activity in vertebrates, ciliates, and yeast. Characterization of the Saccharomyces cerevisiae telomerase RNA (TLC1) pseudoknot identified tertiary structural interactions that are also important for telomerase activity, as previously observed for the Kluyveromyces lactis and human telomerase RNA pseudoknots. In addition, the contributions of backbone ribose 2′-OH groups in the pseudoknot to telomerase catalysis were investigated previously, using 2′-OH (ribose) to 2′-H (deoxyribose) or 2′-O-methyl substitutions in the stem 2 helix, and it was proposed that one or more 2′-OH groups from the stem 2 sequences at or near the triple helix participate in telomerase catalysis. Based on these studies and investigations of the structural and thermodynamic properties of the TLC1 RNA pseudoknot region, we have examined the structural and thermodynamic perturbations of the 2′-O-methyl and 2′-H substituted pseudoknots, using UV-monitored thermal denaturation, native gel electrophoresis, and circular dichroism spectroscopy. Our results demonstrate the presence of A-form helical geometry perturbations in the backbone sugar substituted pseudoknots, show a correlation between thermodynamic stability and telomerase activity, and are consistent with the identification of the U809 ribose 2′-OH as a potential contributor to telomerase activity.     

The impact of dyskeratosis congenita mutations on the structure and dynamics of the human telomerase RNA pseudoknot domain

The pseudoknot domain is a functionally crucial part of telomerase RNA and influences the activity and stability of the ribonucleoprotein complex. Autosomal dominant dyskeratosis congenita (DKC) is an inherited disease that is linked to mutations in telomerase RNA and impairs telomerase function. In this paper, we present a computational prediction of the influence of two base DKC mutations on the structure, dynamics, and stability of the pseudoknot domain. We use molecular dynamics simulations, MM-GBSA free energy calculations, static analysis, and melting simulations analysis. Our results show that the DKC mutations stabilize the hairpin form and destabilize the pseudoknot form of telomerase RNA. Moreover, the P3 region of the predicted DKC-mutated pseudoknot structure is unstable and fails to form as a defined helical stem. We directly compare our predictions with experimental observations by calculating the enthalpy of folding and melting profiles for each structure. The enthalpy values are in very good agreement with values determined by thermal denaturation experiments. The melting simulations and simulations at elevated temperatures show the existence of an intermediate structure, which involves the formation of two UU base pairs observed in the hairpin form of the pseudoknot domain.     

Different role of functional domains of hTR in DNA binding to telomere and telomerase reconstruction

Even if template sequence of hTR played an essential role in telomere binding, a 326 nucleotide fragment of hTR containing template, pseudoknot, and CR4-5 domains is critical for both binding with telomeric DNA and reconstitution of telomerase activity. A functional study with antisense oligonucleotides suggested that targeted disruption of the template region efficiently abrogated both telomeric DNA binding and telomerase activity, whereas disruption of the CR4-5 region induced only loss of telomerase activity. hTR interacts with telomeric DNA via structural region composed of the template, pseudoknot, and CR4-5 domains, however, each structural domain plays a distinct role in telomere binding and telomerase activity reconstitution.