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.     

Entropic stabilization of the folded states of RNA due to macromolecular crowding

We review the effects of macromolecular crowding on the folding of RNA by considering the simplest scenario when excluded volume interactions between crowding particles and RNA dominate. Using human telomerase enzyme as an example, we discuss how crowding can alter the equilibrium between pseudoknot and hairpin states of the same RNA molecule—a key aspect of crowder–RNA interactions. We summarize data showing that the crowding effect is significant only if the size of the spherical crowding particle is smaller than the radius of gyration of the RNA in the absence of crowding particles. The implication for function of the wild type and mutants of human telomerase is outlined by using a relationship between enzyme activity and its conformational equilibrium. In addition, we discuss the interplay between macromolecular crowding and ionic strength of the RNA buffer. Finally, we briefly review recent experiments which illustrate the connection between excluded volume due to macromolecular crowding and the thermodynamics of RNA folding.     

Single-molecule mechanical unfolding and folding of a pseudoknot in human telomerase RNA

RNA unfolding and folding reactions in physiological conditions can be facilitated by mechanical force one molecule at a time. By using force-measuring optical tweezers, we studied the mechanical unfolding and folding of a hairpin-type pseudoknot in human telomerase RNA in a near-physiological solution, and at room temperature. Discrete two-state folding transitions of the pseudoknot are seen at approximately 10 and approximately 5 piconewtons (pN), with ensemble rate constants of approximately 0.1 sec(-1), by stepwise force-drop experiments. Folding studies of the isolated 5'-hairpin construct suggested that the 5'-hairpin within the pseudoknot forms first, followed by formation of the 3'-stem. Stepwise formation of the pseudoknot structure at low forces are in contrast with the one-step unfolding at high forces of approximately 46 pN, at an average rate of approximately 0.05 sec(-1). In the constant-force folding trajectories at approximately 10 pN and approximately 5 pN, transient formation of nonnative structures were observed, which is direct experimental evidence that folding of both the hairpin and pseudoknot takes complex pathways. Possible nonnative structures and folding pathways are discussed.     

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.     

The Impact of Dyskeratosis Congenita Mutations on the Structure and Dynamics of the Human Telomerase RNA Pseudoknot Domain

Abstract

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.     

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.     

Conformational analysis of the telomerase RNA pseudoknot hairpin by Raman spectroscopy

We have measured the temperature-dependent Raman spectra of two 30-mer ribonucleotides that represent the wild-type (WT) and dyskeratosis congenita (DKC) mutant (MT) GC (107-108) --> AG structures of the pseudoknot hairpin region of human telomerase RNA. We have used these structures, previously characterized by UV-melting and NMR, as a model system for our Raman investigation. We observe that Raman hypochromism of vibrational bands, previously assigned to specific bases or conformational RNA markers, reflect temperature-dependent alterations in the pentaloop and stem structures of these two oligonucleotides. We also observe that the intense nu(s)(O-P-O) band at 812 cm(-1) indicates the presence of A-form backbone structure at relatively low temperatures in both the WT and MT RNA sequences. The mutation induces a decrease in the intensity of the uridine (rU) band at 1244 cm(-1) associated with C2'-endo/anti ribose conformation in the pentaloop. Two transition temperatures (T(m) ) were determined from the analysis of Raman difference intensity-temperature profiles of the 1256 cm(-1) band, which is associated with vibrations of cytidine (rC) residues, in particular, the C2'-endo/anti ribose conformation (T(m) 1 = 23.6 +/- 1.6 degrees C for WT and 19.7 +/- 2.8 degrees C for MT; T(m) 2 = 68.9 +/- 1.8 degrees C for WT and 70.9 +/- 1.1 degrees C for MT). From these results we can conclude that the DKC mutant 30-mer exhibits a lower stability in the pentaloop region and a slightly higher stability in the stem region than the WT 30-mer. This demonstrates that Raman bands, previously assigned to specific bases or conformational RNA markers, can be used to probe local structural features of the telomerase pseudoknot hairpin sequence.     

Predicting RNA folding thermodynamics with a reduced chain representation model

Based on the virtual bond representation for the nucleotide backbone, we develop a reduced conformational model for RNA. We use the experimentally measured atomic coordinates to model the helices and use the self-avoiding walks in a diamond lattice to model the loop conformations. The atomic coordinates of the helices and the lattice representation for the loops are matched at the loop-helix junction, where steric viability is accounted for. Unlike the previous simplified lattice-based models, the present virtual bond model can account for the atomic details of realistic three-dimensional RNA structures. Based on the model, we develop a statistical mechanical theory for RNA folding energy landscapes and folding thermodynamics. Tests against experiments show that the theory can give much more improved predictions for the native structures, the thermal denaturation curves, and the equilibrium folding/unfolding pathways than the previous models. The application of the model to the P5abc region of Tetrahymena group I ribozyme reveals the misfolded intermediates as well as the native-like intermediates in the equilibrium folding process. Moreover, based on the free energy landscape analysis for each and every loop mutation, the model predicts five lethal mutations that can completely alter the free energy landscape and the folding stability of the molecule.     

Structure and stability of RNA/RNA kissing complex: with application to HIV dimerization initiation signal

We develop a statistical mechanical model to predict the structure and folding stability of the RNA/RNA kissing-loop complex. One of the key ingredients of the theory is the conformational entropy for the RNA/RNA kissing complex. We employ the recently developed virtual bond-based RNA folding model (Vfold model) to evaluate the entropy parameters for the different types of kissing loops. A benchmark test against experiments suggests that the entropy calculation is reliable. As an application of the model, we apply the model to investigate the structure and folding thermodynamics for the kissing complex of the HIV-1 dimerization initiation signal. With the physics-based energetic parameters, we compute the free energy landscape for the HIV-1 dimer. From the energy landscape, we identify two minimal free energy structures, which correspond to the kissing-loop dimer and the extended-duplex dimer, respectively. The results support the two-step dimerization process for the HIV-1 replication cycle. Furthermore, based on the Vfold model and energy minimization, the theory can predict the native structure as well as the local minima in the free energy landscape. The root-mean-square deviations (RMSDs) for the predicted kissing-loop dimer and extended-duplex dimer are ∼3.0 Å. The method developed here provides a new method to study the RNA/RNA kissing complex.     

Characterization of the mechanical unfolding of RNA pseudoknots

The pseudoknot is an important RNA structural element that provides an excellent model system for studying the contributions of tertiary interactions to RNA stability and to folding kinetics. RNA pseudoknots are also of interest because of their key role in the control of ribosomal frameshifting by viral RNAs. Their mechanical properties are directly relevant to their unfolding by ribosomes during translation. We have used optical tweezers to study the kinetics and thermodynamics of mechanical unfolding and refolding of single RNA molecules. Here we describe the unfolding of the frameshifting pseudoknot from infectious bronchitis virus (IBV), three constituent hairpins, and three mutants of the IBV pseudoknot. All four pseudoknots cause −1 programmed ribosomal frameshifting. We have measured the free energies and rates of mechanical unfolding and refolding of the four frameshifting pseudoknots. Our results show that the IBV pseudoknot requires a higher force than its corresponding hairpins to unfold. Furthermore, its rate of unfolding changes little with increasing force, in contrast with the rate of hairpin unfolding. The presence of Mg²⁺ significantly increases the kinetic barriers to unfolding the IBV pseudoknot, but has only a minor effect on the hairpin unfolding. The greater mechanical stability of pseudoknots compared to hairpins, and their kinetic insensitivity to force supports the hypothesis that −1 frameshifting depends on the difficulty of unfolding the mRNA.     

Dynamic behavior of the telomerase RNA hairpin structure and its relationship to dyskeratosis congenita

In this paper, we present the results from a comprehensive study of nanosecond-scale implicit and explicit solvent molecular dynamics simulations of the wild-type telomerase RNA hairpin. The effects of various mutations on telomerase RNA dynamics are also investigated. Overall, we found that the human telomerase hairpin is a very flexible molecule. In particular, periodically the molecule exhibits dramatic structural fluctuations represented by the opening and closing of a non-canonical base-pair region. These structural deviations correspond to significant disruptions of the direct hydrogen bonding network in the helix, widening of the major groove of the hairpin structure, and causing several U and C nucleotides to protrude into the major groove from the helix permitting them to hydrogen bond with, for example, the P3 domain of the telomerase RNA. We suggest that these structural fluctuations expose a nucleation point for pseudoknot formation. We also found that mutations in the pentaloop and non-canonical region stabilize the hairpin. Moreover, our results show that the hairpin with dyskeratosis congenita mutations is more stable and less flexible than the wild-type hairpin due to base stacking in the pentaloop. The results from our molecular dynamics simulations are in agreement with experimental observations. In addition, they suggest a possible mechanism for pseudoknot formation based on the dynamics of the hairpin structure and also may explain the mutational aspects of dyskeratosis congenita.     

Predicting loop–helix tertiary structural contacts in RNA pseudoknots

Tertiary interactions between loops and helical stems play critical roles in the biological function of many RNA pseudoknots. However, quantitative predictions for RNA tertiary interactions remain elusive. Here we report a statistical mechanical model for the prediction of noncanonical loop–stem base-pairing interactions in RNA pseudoknots. Central to the model is the evaluation of the conformational entropy for the pseudoknotted folds with defined loop–stem tertiary structural contacts. We develop an RNA virtual bond-based conformational model (Vfold model), which permits a rigorous computation of the conformational entropy for a given fold that contains loop–stem tertiary contacts. With the entropy parameters predicted from the Vfold model and the energy parameters for the tertiary contacts as inserted parameters, we can then predict the RNA folding thermodynamics, from which we can extract the tertiary contact thermodynamic parameters from theory–experimental comparisons. These comparisons reveal a contact enthalpy (ΔH) of −14 kcal/mol and a contact entropy (ΔS) of −38 cal/mol/K for a protonated C⁺•(G–C) base triple at pH 7.0, and (ΔH = −7 kcal/mol, ΔS = −19 cal/mol/K) for an unprotonated base triple. Tests of the model for a series of pseudoknots show good theory–experiment agreement. Based on the extracted energy parameters for the tertiary structural contacts, the model enables predictions for the structure, stability, and folding pathways for RNA pseudoknots with known or postulated loop–stem tertiary contacts from the nucleotide sequence alone.     

YNMG tetraloop formation by a dyskeratosis congenita mutation in human telomerase RNA

Autosomal dominant dyskeratosis congenita (DKC) has been linked to mutations in the RNA component of telomerase, the ribonucleoprotein responsible for telomere maintenance. Recent studies have investigated the role of the GC (107-108) --> AG mutation in the conserved P3 helix in the pseudoknot domain of human telomerase RNA. The mutation was found to significantly destabilize the pseudoknot conformation, resulting in a shift in the thermodynamic equilibrium to favor formation of a P2b hairpin intermediate. In the wild-type sequence, the hairpin intermediate was found to form a novel sequence of pyrimidine base pairs in a continuous stem capped by a structured pentaloop. The DKC mutant hairpin was observed to be slightly more stable than the wild-type hairpin, further shifting the pseudoknot-hairpin equilibrium to favor the mutant P2b hairpin. Here we examined the solution structure of the DKC mutant hairpin to identify the reason for this additional stability. We found that the mutant hairpin forms the same stem structure as wild-type and that the additional stabilization observed using optical melting can be explained by the formation of a YNMG-type tetraloop structure, with the last nucleotide of the pentaloop bulged out into the major groove. Our results provide a structural explanation for the increased stability of the mutant hairpin and further our understanding of the effect of this mutation on the structure and stability of the dominant conformation of the pseudoknot domain in this type of DKC.     

Site-specific variations in RNA folding thermodynamics visualized by 2-aminopurine fluorescence

The fluorescent base analogue 2-aminopurine (2-AP) is commonly used to study specific conformational and protein binding events involving nucleic acids. Here, combinations of steady-state and time-resolved fluorescence spectroscopy of 2-AP were employed to monitor conformational transitions within a model hairpin RNA from diverse structural perspectives. RNA substrates adopting stable, unambiguous secondary structures were labeled with 2-AP at an unpaired base, within the loop, or inside the base-paired stem. Steady-state fluorescence was monitored as the RNA hairpins made the transitions between folded and unfolded conformations using thermal denaturation, urea titration, and cation-mediated folding. Unstructured control RNA substrates permitted the effects of higher-order RNA structures on 2-AP fluorescence to be distinguished from stimulus-dependent changes in intrinsic 2-AP photophysics and/or interactions with adjacent residues. Thermodynamic parameters describing local conformational changes were thus resolved from multiple perspectives within the model RNA hairpin. These data provided energetic bases for construction of folding mechanisms, which varied among different folding-unfolding stimuli. Time-resolved fluorescence studies further revealed that 2-AP exhibits characteristic signatures of component fluorescence lifetimes and respective fractional contributions in different RNA structural contexts. Together, these studies demonstrate localized conformational events contributing to RNA folding and unfolding that could not be observed by approaches monitoring only global structural transitions.     

40 Probing single molecule RNA folding using temperature and force

Nucleic acids can be unfolded either by temperature, such as in UV melting, or by mechanical force using optical tweezers. In UV melting experiments, the folding free energy of nucleic acids at mesophilic temperatures are extrapolated from unfolding occurring at elevated temperatures. Additionally, single molecule unfolding experiments are typically performed only at room temperature, preventing calculation of changes in enthalpy and entropy. Here, we present temperature-controlled optical tweezers suitable for studying folding of single RNA molecules at physiological temperatures. Constant temperatures between 22 and 37?°C are maintained with an accuracy of 0.1?°C, whereas the optical tweezers display a spatial resolution of ～1?nm over the temperature range. Using this instrument, we measured the folding thermodynamics and kinetics of a 20-base-pair RNA hairpin by force-ramp and constant force experiments. Between 22 and 37?°C, the hairpin unfolds and refolds in a single step. Increasing temperature decreases the stability of the hairpin and thus decreases the force required to unfold it. The equilibrium force, at which unfolding and refolding rates are equal, drops ～1?pN as temperature increases every 5?°C. At each temperature, the folding energy can be quantified by reversible work done to unfold the RNA and from the equilibrium constant at constant forces. Over the experimental temperature range, the folding free energy of the hairpin depends linearly on temperature, indicating that ΔH is constant. The measured folding thermodynamics are further compared with the nearest neighbor calculations using Turner’s parameters of nucleic acid folding energetics.     

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.     

Prediction of RNA pseudoknots using heuristic modeling with mapping and sequential folding

Predicting RNA secondary structure is often the first step to determining the structure of RNA. Prediction approaches have historically avoided searching for pseudoknots because of the extreme combinatorial and time complexity of the problem. Yet neglecting pseudoknots limits the utility of such approaches. Here, an algorithm utilizing structure mapping and thermodynamics is introduced for RNA pseudoknot prediction that finds the minimum free energy and identifies information about the flexibility of the RNA. The heuristic approach takes advantage of the 5' to 3' folding direction of many biological RNA molecules and is consistent with the hierarchical folding hypothesis and the contact order model. Mapping methods are used to build and analyze the folded structure for pseudoknots and to add important 3D structural considerations. The program can predict some well known pseudoknot structures correctly. The results of this study suggest that many functional RNA sequences are optimized for proper folding. They also suggest directions we can proceed in the future to achieve even better results.