Azoarcus Ribozyme Research Articles

Watching ion-driven kinetics of ribozyme folding and misfolding caused by energetic and topological frustration one molecule at a time.

Folding of ribozymes into well-defined tertiary structures usually requires divalent cations. How Mg2+ ions direct the folding kinetics has been a long-standing unsolved problem because experiments cannot detect the positions and dynamics of ions. To address this problem, we used molecular simulations to dissect the folding kinetics of the Azoarcus ribozyme by monitoring the path each molecule takes to reach the folded state. We quantitatively establish that Mg2+ binding to specific sites, coupled with counter-ion release of monovalent cations, stimulate the formation of secondary and tertiary structures, leading to diverse pathways that include direct rapid folding and trapping in misfolded structures. In some molecules, key tertiary structural elements form when Mg2+ ions bind to specific RNA sites at the earliest stages of the folding, leading to specific collapse and rapid folding. In others, the formation of non-native base pairs, whose rearrangement is needed to reach the folded state, is the rate-limiting step. Escape from energetic traps, driven by thermal fluctuations, occurs readily. In contrast, the transition to the native state from long-lived topologically trapped native-like metastable states is extremely slow. Specific collapse and formation of energetically or topologically frustrated states occur early in the assembly process.

Multispecies autocatalytic RNA reaction networks in coacervates

Robust localization of self-reproducing autocatalytic chemistries is a key step in the realization of heritable and evolvable chemical systems. While autocatalytic chemical reaction networks already possess attributes such as heritable self-reproduction and evolvability, localizing functional multispecies networks within complex primitive phases, such as coacervates, has remained unexplored. Here, we show the self-reproduction of the Azoarcus ribozyme system within charge-rich coacervates where catalytic ribozymes are produced by the autocatalytic assembly of constituent smaller RNA fragments. We systematically demonstrate the catalytic assembly of active ribozymes within phase-separated coacervates—both in micron-sized droplets as well as in a coalesced macrophase, underscoring the facility of the complex, charge-rich phase to support these reactions in multiple configurations. By constructing multispecies reaction networks, we show that these newly assembled molecules are active, participating both in self- and cross-catalysis within the coacervates. Finally, due to differential molecular transport, these phase-separated compartments endow robustness to the composition of the collectively autocatalytic networks against external perturbations. Altogether, our results establish the formation of multispecies self-reproducing reaction networks in phase-separated compartments which in turn render transient robustness to the network composition.

Ion Condensation onto Ribozyme Is Site Specific and Fold Dependent

The highly charged RNA molecules, with each phosphate carrying a single negative charge, cannot fold into well-defined architectures with tertiary interactions in the absence of ions. For ribozymes, divalent cations are known to be more efficient than monovalent ions in driving them to a compact state, although Mg2+ ions are needed for catalytic activities. Therefore, how ions interact with RNA is relevant in understanding RNA folding. It is often thought that most of the ions are territorially and nonspecifically bound to the RNA, as predicted by the counterion condensation theory. Here, we show using simulations of Azoarcus ribozyme, based on an accurate coarse-grained three-site interaction model with explicit divalent and monovalent cations, that ion condensation is highly specific and depends on the nucleotide position. The regions with high coordination between the phosphate groups and the divalent cations are discernible even at very low Mg2+ concentrations when the ribozyme does not form tertiary interactions. Surprisingly, these regions also contain the secondary structural elements that nucleate subsequently in the self-assembly of RNA, implying that ion condensation is determined by the architecture of the folded state. These results are in sharp contrast to interactions of ions (monovalent and divalent) with rigid charged rods, in which ion condensation is uniform and position independent. The differences are explained in terms of the dramatic nonmonotonic shape fluctuations in the ribozyme as it folds with increasing Mg2+ or Ca2+ concentration.

Effects of Preferential Counterion Interactions on the Specificity of RNA Folding.

The real-time search for native RNA structure is essential for the operation of regulatory RNAs. We previously reported that a fraction of the Azoarcus ribozyme achieves a compact structure in less than a millisecond. To scrutinize the forces that drive initial folding steps, we used time-resolved SAXS to compare the folding dynamics of this ribozyme in thermodynamically isostable concentrations of different counterions. The results show that the size of the fast-folding population increases with the number of available counterions and correlates with the flexibility of initial RNA structures. Within 1 ms of folding, Mg2+ exhibits a smaller preferential interaction coefficient per charge, ΔΓ+/ Z, than Na+ or [Co(NH3)6]3+. The lower ΔΓ+/ Z corresponds to a smaller yield of folded RNA, although Mg2+ stabilizes native RNA more efficiently than other ions at equilibrium. These results suggest that strong Mg2+-RNA interactions impede the search for globally native structure during early folding stages.

Spontaneous Covalent Self-Assembly of the Azoarcus Ribozyme from Five Fragments.

Spontaneous covalent assembly of short RNA fragments has been proposed as a plausible prebiotically relevant pathway to a self-reproducing system. We previously showed that the Azoarcus group I intron could self-assemble from four RNA fragments. Here, we extended this fragmentation to five RNAs that averaged <40 nucleotides in length. We optimized this reaction and showed that a dehydration-rehydration sequence was the most effective means to date to shift the self-assembly equilibrium from reactants to products.

Prebiotic RNA Network Formation: A Taxonomy of Molecular Cooperation.

Cooperation is essential for evolution of biological complexity. Recent work has shown game theoretic arguments, commonly used to model biological cooperation, can also illuminate the dynamics of chemical systems. Here we investigate the types of cooperation possible in a real RNA system based on the Azoarcus ribozyme, by constructing a taxonomy of possible cooperative groups. We construct a computational model of this system to investigate the features of the real system promoting cooperation. We find triplet interactions among genotypes are intrinsically biased towards cooperation due to the particular distribution of catalytic rate constants measured empirically in the real system. For other distributions cooperation is less favored. We discuss implications for understanding cooperation as a driver of complexification in the origin of life.

Differential Assembly of Catalytic Interactions within the Conserved Active Sites of Two Ribozymes.

Molecular recognition is central to biology and a critical aspect of RNA function. Yet structured RNAs typically lack the preorganization needed for strong binding and precise positioning. A striking example is the group I ribozyme from Tetrahymena, which binds its guanosine substrate (G) orders of magnitude slower than diffusion. Binding of G is also thermodynamically coupled to binding of the oligonucleotide substrate (S) and further work has shown that the transition from E•G to E•S•G accompanies a conformational change that allows G to make the active site interactions required for catalysis. The group I ribozyme from Azoarcus has a similarly slow association rate but lacks the coupled binding observed for the Tetrahymena ribozyme. Here we test, using G analogs and metal ion rescue experiments, whether this absence of coupling arises from a higher degree of preorganization within the Azoarcus active site. Our results suggest that the Azoarcus ribozyme forms cognate catalytic metal ion interactions with G in the E•G complex, interactions that are absent in the Tetrahymena E•G complex. Thus, RNAs that share highly similar active site architectures and catalyze the same reactions can differ in the assembly of transition state interactions. More generally, an ability to readily access distinct local conformational states may have facilitated the evolutionary exploration needed to attain RNA machines that carry out complex, multi-step processes.

Secondary structure encodes a cooperative tertiary folding funnel in the Azoarcus ribozyme

Secondary structure encodes a cooperative tertiary folding funnel in the Azoarcus ribozyme

Secondary structure encodes a cooperative tertiary folding funnel in the Azoarcus ribozyme.

A requirement for specific RNA folding is that the free-energy landscape discriminate against non-native folds. While tertiary interactions are critical for stabilizing the native fold, they are relatively non-specific, suggesting additional mechanisms contribute to tertiary folding specificity. In this study, we use coarse-grained molecular dynamics simulations to explore how secondary structure shapes the tertiary free-energy landscape of the Azoarcus ribozyme. We show that steric and connectivity constraints posed by secondary structure strongly limit the accessible conformational space of the ribozyme, and that these so-called topological constraints in turn pose strong free-energy penalties on forming different tertiary contacts. Notably, native A-minor and base-triple interactions form with low conformational free energy, while non-native tetraloop/tetraloop–receptor interactions are penalized by high conformational free energies. Topological constraints also give rise to strong cooperativity between distal tertiary interactions, quantitatively matching prior experimental measurements. The specificity of the folding landscape is further enhanced as tertiary contacts place additional constraints on the conformational space, progressively funneling the molecule to the native state. These results indicate that secondary structure assists the ribozyme in navigating the otherwise rugged tertiary folding landscape, and further emphasize topological constraints as a key force in RNA folding.

Kinetic Destabilization through Single Point Mutations Emphasizes the Critical Role Played by Coupled Tertiary Interactions in the Folding of the Azoarcus Ribozyme

Tertiary interactions stabilize RNA structure and impart function to RNAs. Using the Azoarcus group I ribozyme as a model system we determine the contributions of these interactions to folding of RNAs in general. Previous studies have taken a detailed thermodynamic look at key tertiary interactions for this ribozyme [1]. Here we make an effort to unravel the kinetic contributions of these tertiary interactions by abolishing them through single point mutations and studying the resulting dynamics through smFRET. We see that tertiary interaction mutants promote an increased number of excursions from the compact folded state of the ribozyme along with broadening the FRET population distribution. Our results show that the change in folding kinetics for each of the mutants is context dependent, providing us a kinetic stability map of the ribozyme's key tertiary interactions. A mutation in the P9 tetraloop results in the most “floppy” ribozyme, while a mutation in the P2 tetraloop causes only a small increase in transient unfolding, compared to the wild type ribozyme. Among the mutants, the folding rates show a smaller variation and are faster than the unfolding rates. These results show us how this long noncoding RNA has evolved to reduce conformational fluctuations as it proceeds in its folding path towards the native state.[1] Behrouzi R., Roh J.H., Kilburn D., Briber R.M., Woodson S.A.; Cell, 2012, 149, 2, 348-357

Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme.

The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg2+ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg2+ concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.

A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction.

Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the binding-competent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5′-splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5′-exon intermediate in self splicing to remain bound subsequent to 5′-exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs.

Development and Applications of Coarse‐Grained Models for RNA

AbstractIn contrast to proteins, much less attention has been focused on the development of computational models for describing RNA molecules, which are being recognized as playing key roles in many cellular functions. Current atomically detailed force fields are not accurate enough to capture the properties of even simple nucleic acid constructs. In this article, we review our efforts to develop coarse‐grained (CG) models that capture the underlying physics for the particular length scale of interest. Two models are discussed. One of them is the three interaction site (TIS) model, in which each nucleotide is represented by three beads corresponding to sugar, phosphate, and base. The other is the self‐organized polymer (SOP) model, in which each nucleotide is represented as a single interaction center. Applications of the TIS model to study the complexity of hairpin formation and the effects of crowding in a shifting equilibrium between two conformations in human telomerase pseudoknot are described. The work on crowding illustrates a direct link to the activity of telomerase. We use the SOP model to describe the response of the Tetrahymena ribozyme to force. The simulated unfolding pathways agree well with single molecule pulling experiments. We also review predictions for the unfolding pathways for the Azoarcus ribozyme. The success of the CG applications to describe dynamics in RNA gives hope that more complex processes involving RNA‐protein interactions can be tackled using variants of the proposed models.

Trans-splicing with the group I intron ribozyme from Azoarcus

Group I introns are ribozymes (catalytic RNAs) that excise themselves from RNA primary transcripts by catalyzing two successive transesterification reactions. These cis-splicing ribozymes can be converted into trans-splicing ribozymes, which can modify the sequence of a separate substrate RNA, both in vitro and in vivo. Previous work on trans-splicing ribozymes has mostly focused on the 16S rRNA group I intron ribozyme from Tetrahymena thermophila. Here, we test the trans-splicing potential of the tRNA(Ile) group I intron ribozyme from the bacterium Azoarcus. This ribozyme is only half the size of the Tetrahymena ribozyme and folds faster into its active conformation in vitro. Our results showed that in vitro, the Azoarcus and Tetrahymena ribozymes favored the same set of splice sites on a substrate RNA. Both ribozymes showed the same trans-splicing efficiency when containing their individually optimized 5' terminus. In contrast to the previously optimized 5'-terminal design of the Tetrahymena ribozyme, the Azoarcus ribozyme was most efficient with a trans-splicing design that resembled the secondary structure context of the natural cis-splicing Azoarcus ribozyme, which includes base-pairing between the substrate 5' portion and the ribozyme 3' exon. These results suggested preferred trans-splicing interactions for the Azoarcus ribozyme under near-physiological in vitro conditions. Despite the high activity in vitro, however, the splicing efficiency of the Azoarcus ribozyme in Escherichia coli cells was significantly below that of the Tetrahymena ribozyme.

Recycling of Informational Units Leads to Selection of Replicators in a Prebiotic Soup

Prebiotic chemical reactions would have been greatly aided by a process whereby living materials could have been recycled under conditions of limiting resources. Recombination of RNA fragments is a viable means of recycling but has not been demonstrated. Using systems based on the Azoarcus group I intron ribozyme, computational Monte Carlo studies indicate that a moderate level of recycling activity, spontaneous or catalyzed, leads to the most robust selection scenarios. It is interesting that recycling leads to a threshold effect wherea dominant species suddenly jumps to fixation. In conjunction, laboratory studies with the Azoarcus ribozyme corroborate these results, showing that mixtures of scrambled and/or deleteriously mutated molecules can recycle their component fragments to generate fully functional recombinase ribozymes. These studies highlight the importance of recombination and recycling jointly in the advent of living systems.

Counterion-Dependent Folding of Azoarcus Ribozyme by SAXS Profile based Hybrid Simulation

Multivalent counterions are essential for self-assembly processes of many functional RNAs. With increasing counterion concentrations, RNAs adopt compact intermediates and finally reach their native states. While the changes in global features such as molecular size and flexibility are often discussed using small angle X-ray scattering (SAXS) data, further details of the processes are difficult to address. Here, by sampling the conformational ensemble of the Azoarcus ribozyme using self-organized polymer model, we obtained the structural ensemble of the molecule that confirmed by a pair distance distribution function at various Mg2+ ion concentrations. At each Mg2+ ion condition, there could be many different structural ensembles, each of which equally reproduces the same SAXS profile. Hence, we carried out clustering analysis on the molecular ensembles by using the contact matrices defined for six different paired domains in secondary structure, and analyzed how the Azoarcus ribozyme reaches its native conformation by quantifying the similarity between the clusters. In consistent with the kinetic partitioning mechanism theory, the folding process of the Azoarcus ribozyme adopts multiple pathway. These pathways are more diverse above the transition mid-concentration of Mg2+ ions, suggesting that there are many intermediates along the folding process. Interestingly, although urea shifts the Mg2+ concentration of folding transition to a higher value, the dominant population of folding pathway is formed along the clusters with larger native contacts. This shows that urea can anneal the pathways of the Azoarcus folding dynamics towards the native state.

The Role of Electrostatic Relaxation on the Folding Kinetics of a Bacterial Ribozyme

The self-assembly of catalytic RNA ribozymes into compact, native structures is critical for their functions in the cell. The first step in forming RNA tertiary structure is the neutralization by cations of the negative charges of the phosphates. This electrostatic stabilization enables dynamical exploration of more compact conformations, and the search for long-range tertiary interactions. Our previous time-resolved Small Angle X-ray Scattering (SAXS) studies showed that the Azoarcus ribozyme exhibits triphasic folding kinetics: up to 90% folds in less than 10 ms, which we attribute to specific collapse.

An all RNA Hypercyclic Network

A hypercycle is a cooperative organization of autocatalytic units that are associated in a cyclic linkage in such a way that there is a mutual survival and regulated growth of all the units involved. Compared to non‐coupled self‐replicating units, which can only sustain a limited amount of genetic information, the hypercycle allows the maintenance of large amounts of information through cooperation among otherwise competitive units. As such, this reaction network may have played a significant role in precellular evolution during the origins of life on the Earth. However, hypercycles have never been empirically demonstrated in the absence of cell‐like compartmentalization. In the current work, hypercyclic behavior is demonstrated in the autocatalytic assembly of Azoarcus group I ribozyme. Three different constructs of the Azoarcus ribozyme with different internal guide sequences (IGS) – GUG (canonical), GAG, and GCG – are capable of a minimal amount of self‐assembly when broken into two fragments. Here, self‐assembly depends on a mismatch with non‐complementary sequences, CGU, CAU and CUU, respectively, to be recognized by IGS via autocatalysis. Yet when all three constructs are present in the same reaction vessel, concomitant assembly of all three is enhanced through an interdependent hypercyclic reaction network. Analysis of these reactions indicates that each system is capable of guiding its own reproduction weakly, along with providing enhanced catalytic support for the reproduction of one other construct system through matched IGS‐tag interactions. Also, when co‐incubated with non‐interacting (i.e., selfish) yet efficient self‐assembly systems, the hypercyclic assembly outcompetes the selfish self‐assembly systems, demonstrating the ability of a hypercyclic organization to possess an evolutionary advantage.

Time-Resolved Multiphase Folding of Bacterial Group I Ribozyme

Understanding of microscopic mechanism of the RNA folding process is critical for mapping out the complex mechanisms by which RNA mediates various cellular events including gene-information transfer and catalysis. Folding of RNA occurs in the presence of cations that neutralize the anionic charge of the phosphate groups, and subsequently enable long distance tertiary interactions between nucleotide bases. The process by which RNA rapidly finds its unique three-dimensional shape despite countless possible conformations remains an unsolved problem. We aim to understand the roles of electrostatic neutralization and cooperative tertiary interactions in the kinetic nucleation of structure and collapse. We have used time-resolved small angle X-ray scattering (SAXS) coupled with a stopped-flow sample system that covers sub-ms timescales to monitor the real-time structural change in the Azoarcus ribozyme. The collapse process was found to have at least two distinct kinetic phases within 100 ms. As the concentration of cations increases, both the kinetic constant (k) and fraction of fast collapsing phase increase up to k ∼ ≥ 3000 s−1 and 80 % for high Mg2+ concentrations and from ∼ 2000 s−1 and 40 % for low Mg2+ concentrations. This suggests that the initial charge-screening rate is crucial for determining not only the kinetic constant for the formation of tertiary interactions but also the partitioning into faster pathways. A minority (∼10%) of the Azoarcus ribozyme falls into slow collapsing pathways, with k ∼ 0.03 s−1. The results imply that the multiphase collapse process originates from an ensemble of heterogeneous structures folding on different pathways rather than a sequential step-like process. Strong kinetic heterogeneity of the collapse nucleation appeared near the thermodynamic transition, which suggests a strong competition among energetically different collapse pathways.

Metal Ion Dependence of Cooperative Collapse Transitions in RNA

Positively charged counterions drive RNA molecules into compact configurations that lead to their biologically active structures. To understand how the valence and size of the cations influences the collapse transition in RNA, small-angle X-ray scattering was used to follow the decrease in the radius of gyration ( R g) of the Azoarcus and Tetrahymena ribozymes in different cations. Small, multivalent cations induced the collapse of both ribozymes more efficiently than did monovalent ions. Thus, the cooperativity of the collapse transition depends on the counterion charge density. Singular value decomposition of the scattering curves showed that folding of the smaller and more thermostable Azoarcus ribozyme is well described by two components, whereas collapse of the larger Tetrahymena ribozyme involves at least one intermediate. The ion-dependent persistence length, extracted from the distance distribution of the scattering vectors, shows that the Azoarcus ribozyme is less flexible at the midpoint of transition in low-charge-density ions than in high-charge-density ions. We conclude that the formation of sequence-specific tertiary interactions in the Azoarcus ribozyme overlaps with neutralization of the phosphate charge, while tertiary folding of the Tetrahymena ribozyme requires additional counterions. Thus, the stability of the RNA structure determines its sensitivity to the valence and size of the counterions.