Folding of human telomerase RNA pseudoknot using ion-jump and temperature-quench simulations

Globally RNA folding occurs in multiple stages involving chain compaction and subsequent rearrangement by a number of parallel routes to the folded state. However, the sequence-dependent details of the folding pathways and the link between collapse and folding are poorly understood. To obtain a comprehensive picture of the thermodynamics and folding kinetics we used molecular simulations of coarse-grained model of a pseudoknot found in the conserved core domain of the human telomerase (hTR) by varying both temperature (T) and ion concentration (C). The phase diagram in the [T,C] plane shows that the boundary separating the folded and unfolded state for the finite 47-nucleotide system is relatively sharp, implying that from a thermodynamic perspective hTR behaves as an apparent two-state system. However, the folding kinetics following single C-jump or T-quench is complicated, involving multiple channels to the native state. Although globally folding kinetics triggered by T-quench and C-jump are similar, the kinetics of chain compaction are vastly different, which reflects the role of initial conditions in directing folding and collapse. Remarkably, even after substantial reduction in the overall size of hTR, the ensemble of compact conformations are far from being nativelike, suggesting that the search for the folded state occurs among the ensemble of low-energy fluidlike globules. The rate of unfolding, which occurs in a single step, is faster upon C-decrease compared to a jump in temperature. To identify "hidden" states that are visited during the folding process we performed simulations by periodically interrupting the approach to the folded state by lowering C. These simulations show that hTR reaches the folded state through a small number of connected clusters that are repeatedly visited during the pulse sequence in which the folding or unfolding is interrupted. The results from interrupted folding simulations, which are in accord with non-equilibrium single-molecule folding of a large ribozyme, show that multiple probes are needed to reveal the invisible states that are sampled by RNA as it folds. Although we have illustrated the complexity of RNA folding using hTR as a case study, general arguments and qualitative comparisons to time-resolved scattering experiments on Azoarcus group I ribozyme and single-molecule non-equilibrium periodic ion-jump experiments establish the generality of our findings.     

Biologically important reactions catalyzed by RNA molecules

The last few years have seen a considerable increase in our understanding of catalysis by naturally occurring RNA molecules called ribozymes. The biological functions of RNA molecules depend upon their adoption of appropriate three-dimensional structures. The structure of RNA has a very important electrostatic component, which results from the presence of charged phosphodiester bonds. Metal ions are usually required to stabilize the folded structures and/or catalysis. Some ribozymes utilize metal ions as catalysts, whereas others use the ions to maintain appropriate three-dimensional structures. In the latter case, the correct folding of the RNA structures can perturb the pKa values of the nucleotide(s) within a catalytic pocket such that they act as general acid/bases catalysts.     

Atomistic analysis of pseudoknotted RNA unfolding

As important functional structures, RNA pseudoknots provide excellent models for studying the interplay between secondary and tertiary structures and the roles of triplexes, noncanonical interactions, and coaxial stacking in the folding/unfolding process. Here we report a first atomistic and statistical analysis of the unfolding of the pseudoknot within gene 32 mRNA of bacteriophage T2. Multiple unfolding pathways, diverse transition states, and various intermediate structures were observed. Water molecules were found to be coupled with the unfolding process via the expulsion or concurrent mechanism.     

The shape-shifting quasispecies of RNA: one sequence, many functional folds

E Unus pluribum, or "Of One, Many", may be at the root of decoding the RNA sequence-structure-function relationship. RNAs embody the large majority of genes in higher eukaryotes and fold in a sequence-directed fashion into three-dimensional structures that perform functions conserved across all cellular life forms, ranging from regulating to executing gene expression. While it is the most important determinant of the RNA structure, the nucleotide sequence is generally not sufficient to specify a unique set of secondary and tertiary interactions due to the highly frustrated nature of RNA folding. This frustration results in folding heterogeneity, a common phenomenon wherein a chemically homogeneous population of RNA molecules folds into multiple stable structures. Often, these alternative conformations constitute misfolds, lacking the biological activity of the natively folded RNA. Intriguingly, a number of RNAs have recently been described as capable of adopting multiple distinct conformations that all perform, or contribute to, the same function. Characteristically, these conformations interconvert slowly on the experimental timescale, suggesting that they should be regarded as distinct native states. We discuss how rugged folding free energy landscapes give rise to multiple native states in the Tetrahymena Group I intron ribozyme, hairpin ribozyme, sarcin-ricin loop, ribosome, and an in vitro selected aptamer. We further describe the varying degrees to which folding heterogeneity impacts function in these RNAs, and compare and contrast this impact with that of heterogeneities found in protein folding. Embracing that one sequence can give rise to multiple native folds, we hypothesize that this phenomenon imparts adaptive advantages on any functionally evolving RNA quasispecies.     

Long RNA dangling end has large energetic contribution to duplex stability

Long terminal unpaired nucleotides known as dangling ends play interesting roles in biological systems. Previous studies, however, only dealt with the energy contributions of single dangling bases. The energy contributions of long dangling ends on the stability of duplexes have not been systematically studied. We now report a quantitative increase in stability of RNA-RNA and DNA-DNA duplexes containing a long dangling end. We found a larger enhancement of the stability by the long RNA dangling end of the RNA-RNA duplex than has been observed for the DNA duplexes. It is also found that structural stabilizations by long dangling ends seem to originate from the single-stranded stacking interactions of nucleotides. These results indicate that RNA stability can be achieved by increasing the length of the dangling end. The thermodynamic parameters of the long dangling ends are useful for designing ribozymes and antisense oligonucleotides, and for the prediction of the RNA secondary structure like the pseudoknot.     

Direct measurement of tertiary contact cooperativity in RNA folding

All structured biological macromolecules must overcome the thermodynamic folding problem to populate a unique functional state among a vast ensemble of unfolded and alternate conformations. The exploration of cooperativity in protein folding has helped reveal and distinguish the underlying mechanistic solutions to this folding problem. Analogous dissections of RNA tertiary stability remain elusive, however, despite the central biological importance of folded RNA molecules and the potential to reveal fundamental properties of structured macromolecules via comparisons of protein and RNA folding. We report a direct quantitative measure of tertiary contact cooperativity in a folded RNA. We precisely measured the stability of an independently folding P4-P6 domain from the Tetrahymena thermophila group I intron by single molecule fluorescence resonance energy transfer (smFRET). Using wild-type and mutant RNAs, we found that cooperativity between the two tertiary contacts enhances P4-P6 stability by 3.2 +/- 0.2 kcal/mol.     

In vitro selection of a viomycin-binding RNA pseudoknot

Background: The peptide antibiotic viomycin inhibits ribosomal protein synthesis, group I intron self-splicing and self-cleavage of the human hepatitis delta virus ribozyme. To understand the molecular basis of RNA binding and recognition by viomycin, we isolated a variety of novel viomycin-binding RNA molecules using in vitro selection.Results: More than 90% of the selected RNA molecules shared one continuous highly conserved region of 14 nucleotides. Mutational analyses, structural probing, together with footprinting experiments by chemical modification, and Pb²⁺-induced cleavage showed that this conserved sequence harbours the antibiotic-binding site and forms a stem-loop structure. Moreover, the loop is engaged in a long-range interaction forming a pseudoknot.Conclusions: A comparison between the novel viomycin-binding motif and the natural RNA target sites for viomycin showed that all these segments form a pseudoknot at the antibiotic-binding site. We therefore conclude that this peptide antibiotic has a strong selectivity for particular RNA pseudoknots.     

Structure and Function of the Ribosomal Frameshifting Pseudoknot RNA from Beet Western Yellow Virus

Many viruses reprogram ribosomes to produce two different proteins from two different reading frames. So‐called ?1 frameshifting often involves pairwise alignment of two adjacent tRNAs at a ‘slippery' sequence in the ribosomal A and P sites such that an overlapping codon is shifted upstream by one base relative to the zero frame. In the majority of cases, an RNA pseudoknot located downstream stimulates this type of frameshift. Crystal structures of the frameshifting RNA pseudoknot from Beet Western Yellow Virus (BWYV) have provided a detailed picture of the tertiary interactions stabilizing this folding motif, including a minor‐groove triplex and quadruple‐base interactions. The structure determined at atomic resolution revealed the locations of several magnesium ions and provided insights into the role of structured water stabilizing the RNA. Systematic in vitro and in vivo mutational analyses based on the structural results revealed specific tertiary interactions and regions in the pseudoknot that drastically change frameshifting efficiency. Here, we summarize recent advances in our understanding of pseudoknot‐mediated ribosomal frameshifting on the basis of the insights gained from structural and functional studies of the RNA pseudoknot from BWYV.     

Pseudoknot preorganization of the preQ1 class I riboswitch

To explore folding and ligand recognition of metabolite-responsive RNAs is of major importance to comprehend gene regulation by riboswitches. Here, we demonstrate, using NMR spectroscopy, that the free aptamer of a preQ(1) class I riboswitch preorganizes into a pseudoknot fold in a temperature- and Mg(2+)-dependent manner. The preformed pseudoknot represents a structure that is close to the ligand-bound state and that likely represents the conformation selected by the ligand. Importantly, a defined base pair mutation within the pseudoknot interaction stipulates whether, in the absence of ligand, dimer formation of the aptamer competes with intramolecular pseudoknot formation. This study pinpoints how RNA preorganization is a crucial determinant for the adaptive recognition process of RNA and ligand.     

Non‐B DNA Structures Show Diverse Conformations and Complex Transition Kinetics Comparable to RNA or Proteins—A Perspective from Mechanical Unfolding and Refolding Experiments

With the firm demonstration of the in vivo presence and biological functions of many non‐B DNA structures, it is of great significance to understand their physiological roles from the perspective of structural conformation, stability, and transition kinetics. Although relatively simple in primary sequences compared to proteins, non‐B DNA species show rather versatile conformations and dynamic transitions. As the most‐studied non‐B DNA species, the G‐quadruplex displays a myriad of conformations that can interconvert between each other in different solutions. These features impose challenges for ensemble‐average techniques, such as X‐ray crystallography, NMR spectroscopy, and circular dichroism (CD), but leave room for single‐molecular approaches to illustrate the structure, stability, and transition kinetics of individual non‐B DNA species in a solution mixture. Deconvolution of the mixture can be further facilitated by statistical data treatment, such as iPoDNano (i ntegrated po pulation d econvolution with nano meter resolution), which resolves populations with subnanometer size differences. This Personal Account summarizes current mechanical unfolding and refolding methods to interrogate single non‐B DNA species, with an emphasis on DNA G‐quadruplexes and i‐motifs. These single‐molecule studies start to demonstrate that structures and transitions in non‐B DNA species can approach the complexity of those in RNA or proteins, which provides solid justification for the biological functions carried out by non‐B DNA species.     

Generation and development of RNA ligase ribozymes with modular architecture through "design and selection"

In vitro selection with long random RNA libraries has been used as a powerful method to generate novel functional RNAs, although it often requires laborious structural analysis of isolated RNA molecules. Rational RNA design is an attractive alternative to avoid this laborious step, but rational design of catalytic modules is still a challenging task. A hybrid strategy of in vitro selection and rational design has been proposed. With this strategy termed "design and selection," new ribozymes can be generated through installation of catalytic modules onto RNA scaffolds with defined 3D structures. This approach, the concept of which was inspired by the modular architecture of naturally occurring ribozymes, allows prediction of the overall architectures of the resulting ribozymes, and the structural modularity of the resulting ribozymes allows modification of their structures and functions. In this review, we summarize the design, generation, properties, and engineering of four classes of ligase ribozyme generated by design and selection.     

二维蛋白质模型分子在折叠过程中的构象研究

蛋白质在折叠过程中其构象要发生明显变化 .采用精确计数法 ,计算了在蛋白质折叠的不同阶段其尺寸大小及其分布情况 .发现在折叠的初期 ,分子的尺寸比较大 ,其分布也比较宽 .在折叠的后期 ,分子的尺寸比较小 ,其分布比较窄 .不同的氨基酸序列 ,其分子尺寸的分布也不同 .对于可折叠的氨基酸序列 ,在平均尺寸大小附近出现的几率特别大 .同时还计算了比值SN DN.这里SN 为可设计序列数目 (Thenumberofdesigningsequences) ,DN 为可设计构象数目 (Thenumberofdesignableconformations) ,并有关系- 1 6 8 4 +0 32 5 5N≤SN DN ≤- 0 86 6 4 +0 312 5N　 (N ≥ 13)通过这些研究以提高对折叠过程的认识     

A practical valence bond method: a configuration interaction method approach with perturbation theoretic facility

The previously developed valence bond configuration interaction (VBCI) method (Wu, W.; Song, L.; Cao, Z.; Zhang, Q.; Shaik, S., J. Phys. Chem. A, 2002, 105, 2721) that borrows the general CI philosophy of the MO theory, is further extended in this article, and its methodological features are improved, resulting in three accurate and cost-effective procedures: (a) the effect of quadruplet excitation is incorporated using the Davidson correction, such that the new procedure reduces size consistency problems, with due improvement in the quality of the computational results. (b) A cost-effective procedure, named VBCI(D, S), is introduced. It includes doubly excited structures for active electrons and singly excited structures for inactive pairs. The computational results of VBCI(D, S) match those of VBCISD with much less computational effort than VBCISD. (c) Finally, a second-order perturbation theory is utilized as a means of configuration selection, and lead to considerable reduction of the computational cost, with little or no loss in accuracy. Applications of the new procedures to bond energies and barriers of chemical reactions are presented and discussed.     

Ligand requirements for glmS ribozyme self-cleavage

Natural RNA catalysts (ribozymes) perform essential reactions in biological RNA processing and protein synthesis, whereby catalysis is intrinsic to RNA structure alone or in combination with metal ion cofactors. The recently discovered glmS ribozyme is unique in that it functions as a glucosamine-6-phosphate (GlcN6P)-dependent catalyst believed to enable "riboswitch" regulation of amino-sugar biosynthesis in certain prokaryotes. However, it is unclear whether GlcN6P functions as an effector or coenzyme to promote ribozyme self-cleavage. Herein, we demonstrate that ligand is absolutely requisite for glmS ribozyme self-cleavage activity. Furthermore, catalysis both requires and is dependent upon the acid dissociation constant (pKa) of the amine functionality of GlcN6P and related compounds. The data demonstrate that ligand is integral to catalysis, consistent with a coenzyme role for GlcN6P and illustrating an expanded capacity for biological RNA catalysis.     

Repurposing Antiviral Drugs for Orthogonal RNA‐Catalyzed Labeling of RNA

In vitro selected ribozymes are promising tools for site‐specific labeling of RNA. Previously known nucleic acid catalysts attached fluorescently labeled adenosine or guanosine derivatives through 2′,5′‐branched phosphodiester bonds to the RNA of interest. Herein, we report new ribozymes that use orthogonal substrates, derived from the antiviral drug tenofovir, and attach bioorthogonal functional groups, as well as affinity handles and fluorescent reporter units through a hydrolytically more stable phosphonate ester linkage. The tenofovir transferase ribozymes were identified by in vitro selection and are orthogonal to nucleotide transferase ribozymes. As genetically encodable functional RNAs, these ribozymes may be developed for potential cellular applications. The orthogonal ribozymes addressed desired target sites in large RNAs in vitro, as shown by fluorescent labeling of E. coli 16S and 23S rRNAs in total cellular RNA.     

Repurposing Antiviral Drugs for Orthogonal RNA-Catalyzed Labeling of RNA

In vitro selected ribozymes are promising tools for site-specific labeling of RNA. Previously known nucleic acid catalysts attached fluorescently labeled adenosine or guanosine derivatives through 2′,5′-branched phosphodiester bonds to the RNA of interest. Herein, we report new ribozymes that use orthogonal substrates, derived from the antiviral drug tenofovir, and attach bioorthogonal functional groups, as well as affinity handles and fluorescent reporter units through a hydrolytically more stable phosphonate ester linkage. The tenofovir transferase ribozymes were identified by in vitro selection and are orthogonal to nucleotide transferase ribozymes. As genetically encodable functional RNAs, these ribozymes may be developed for potential cellular applications. The orthogonal ribozymes addressed desired target sites in large RNAs in vitro, as shown by fluorescent labeling of E. coli 16S and 23S rRNAs in total cellular RNA.     

Exploring the energy landscape of a small RNA hairpin

The energy landscape of a small RNA tetraloop hairpin is explored by temperature jump kinetics and base-substitution. The folding kinetics are single-exponential near the folding transition midpoint T(m). An additional fast phase appears below the midpoint, and an additional slow phase appears above the midpoint. Stem mutation affects the high-temperature phase, while loop mutation affects the low-temperature phase. An adjusted 2-D lattice model reproduces the temperature-dependent phases, although it oversimplifies the structural interpretation. A four-state free energy landscape model is generated based on the lattice model. This model explains the thermodynamics and multiphase kinetics over the full temperature range of the experiments. An analysis of three variants shows that one of the intermediate RNA structures is a stacking-related trap affected by stem but not loop modification, while the other is an early intermediate that forms some stem and loop structure. Even a very fast-folding 8-mer RNA with an ideal tetraloop sequence has a rugged energy landscape, ideal for testing analytical and computational models.     

Structural basis of telomeric RNA quadruplex--acridine ligand recognition

Human telomeric DNA is now known to be transcribed into noncoding RNA sequences, termed TERRA. These sequences, which are believed to play roles in the regulation of telomere function, can form higher-order quadruplex structures and may themselves be the target of therapeutic intervention. The crystal structure of a TERRA quadruplex-acridine small-molecule complex at a resolution of 2.60 ?, is reported here and contrasts remarkably with the structure of the analogous DNA quadruplex complex. The bimolecular RNA complex has a parallel-stranded topology with propeller-like UUA loops. These loops are held in particular conformations by multiple hydrogen bonds involving the O2' hydroxyl groups of the ribonucleotide sugars and play an active role in binding the acridine molecules to the RNA quadruplex. By contrast, the analogous DNA quadruplex complex has simpler 1:1 acridine binding, with no loop involvement. There are significant loop conformational changes in the RNA quadruplex compared to the native TERRA quadruplex (Collie, G. W.; Haider, S. M.; Neidle, S.; Parkinson, G. N. Nucleic Acids Res. 2010, 38, 5569 - 5580), which have implications for the future design of small molecules targeting TERRA quadruplexes, and RNA quadruplexes more generally.     

Conformational dynamics of bistable RNAs studied by time-resolved NMR spectroscopy

The structural transition between two alternate conformations of bistable RNAs has been characterized by time-resolved NMR spectroscopy. The mechanism, kinetics, and thermodynamics underlying the global structural transition of bistable RNAs were delineated. Both bistable RNA conformations and a partial unstructured RNA of identical sequence could be trapped using photolabile protecting groups. This trapping allowed for an investigation of the initial folding from an unfolded RNA to one of the preferred conformations of the bistable RNA and of the structural transitions involved. Folding of the secondary structure elements occurs rapidly, while the global structural transition of the bistable RNA occurs on a time scale of minutes and shows marked temperature dependence. Comparison of these results with bistable systems previously investigated leads to the prediction of activation enthalpies (DeltaH++) associated with global structural transitions in RNA.