Investigation of adenosine base ionization in the hairpin ribozyme by nucleotide analog interference mapping.

Tertiary structure in globular RNA folds can create local environments that lead to pKa perturbation of specific nucleotide functional groups. To assess the prevalence of functionally relevant adenosine-specific pKa perturbation in RNA structure, we have altered the nucleotide analog interference mapping (NAIM) approach to include a series of a phosphorothioate-tagged adenosine analogs with shifted N1 pKa values. We have used these analogs to analyze the hairpin ribozyme, a small self-cleaving/ligating RNA catalyst that is proposed to employ a general acid-base reaction mechanism. A single adenosine (A10) within the ribozyme active site displayed an interference pattern consistent with a functionally significant base ionization. The exocyclic amino group of a second adenosine (A38) contributes substantially to hairpin catalysis, but ionization of the nucleotide does not appear to be important for activity. Within the hairpin ribozyme crystal structure, A10 and A38 line opposite edges of a solvent-excluded cavity adjacent to the 5'-OH nucleophile. The results are inconsistent with the model of ribozyme chemistry in which A38 acts as a general acid-base catalyst, and suggest that the hairpin ribozyme uses an alternative mechanism to achieve catalytic rate enhancement that utilizes functional groups within a solvent-excluded cleft in the ribozyme active site.     

Nucleotide analog interference mapping of the hairpin ribozyme: implications for secondary and tertiary structure formation.

The hairpin ribozyme is a small, naturally occurring RNA capable of folding into a distinct three-dimensional structure and catalyzing a specific phosphodiester transfer reaction. We have adapted a high throughput screening procedure entitled nucleotide analog interference mapping (NAIM) to identify functional groups important for proper folding and catalysis of this ribozyme. A total of 18 phosphorothioate-tagged nucleotide analogs were used to determine the contribution made by individual ribose 2'-OH and purine functional groups to the hairpin ribozyme ligation reaction. Substitution with 2'-deoxy-nucleotide analogs disrupted activity at six sites within the ribozyme, and a unique interference pattern was observed at each of the 11 conserved purine nucleotides. In most cases where such information is available, the NAIM data agree with the previously reported single-site substitution results. The interference patterns are interpreted in comparison to the isolated loop A and loop B NMR structures and a model of the intact ribozyme. These data provide biochemical evidence in support of many, but not all, of the non-canonical base-pairs observed by NMR in each loop, and identify the functional groups most likely to participate in the tertiary interface between loop A and loop B. These groups include the 2'-OH groups of A10, G11, U12, C25, and A38, the exocyclic amine of G11, and the minor groove edge of A9 and A24. The data also predict non-A form sugar pucker geometry at U39 and U41. Based upon these results, a revised model for the loop A tertiary interaction with loop B is proposed. This work defines the chemical basis of purine nucleotide conservation in the hairpin ribozyme, and provides a basis for the design and interpretation of interference suppression experiments.     

Distinct modes of mature and precursor tRNA binding to Escherichia coli RNase P RNA revealed by NAIM analyses

We have analyzed by nucleotide analog interference mapping (NAIM) pools of precursor or mature tRNA molecules, carrying a low level of Rp-RMPalphaS (R = A, G, I) or Rp-c7-deaza-RMPalphaS (R = A, G) modifications, to identify functional groups that contribute to the specific interaction with and processing efficiency by Escherichia coli RNase P RNA. The majority of interferences were found in the acceptor stem, T arm, and D arm, including the strongest effects observed at positions G19, G53, A58, and G71. In some cases (interferences at G5, G18, and G71), the affected functional groups are candidates for direct contacts with RNase P RNA. Several modifications disrupt intramolecular tertiary contacts known to stabilize the authentic tRNA fold. Such indirect interference effects were informative as well, because they allowed us to compare the structural constraints required for ptRNA processing versus product binding. Our ptRNA processing and mature tRNA binding NAIM analyses revealed overlapping but nonidentical patterns of interference effects, suggesting that substrate binding and cleavage involves binding modes or conformational states distinct from the binding mode of mature tRNA, the product of the reaction.     

Comparative analysis of hairpin ribozyme structures and interference data

Great strides in understanding the molecular underpinnings of RNA catalysis have been achieved with advances in RNA structure determination by NMR spectroscopy and X-ray crystallography. Despite these successes the functional relevance of a given structure can only be assessed upon comparison with biochemical studies performed on functioning RNA molecules. The hairpin ribozyme presents an excellent case study for such a comparison. The active site is comprised of two stems each with an internal loop that forms a series of non-canonical base pairs. These loops dock into each other to create an active site for catalysis. Recently, three independent structures have been determined for this catalytic RNA, including two NMR structures of the isolated loop A and loop B stems and a high-resolution crystal structure of both loops in a docked conformation. These structures differ significantly both in their tertiary fold and the nature of the non-canonical base pairs formed within each loop. Several of the chemical groups required to achieve a functioning hairpin ribozyme have been determined by nucleotide analog interference mapping (NAIM). Here we compare the three hairpin structures with previously published NAIM data to assess the convergence between the structural and functional data. While there is significant disparity between the interference data and the individual NMR loop structures, there is almost complete congruity with the X-ray structure. The only significant differences cluster around an occluded pocket adjacent to the scissile phosphate. These local differences may suggest a role for these atoms in the transition state, either directly in chemistry or via a local structural rearrangement.     

Quantitation of free energy profiles in RNA-ligand interactions by nucleotide analog interference mapping

RNA interactions with protein and small molecule ligands serve a wide variety of biochemical functions in the cell. To best understand the specificity and affinity of these interactions, the free energy contribution made by individual function groups in the RNA must be determined. As an efficient method for obtaining such energetic profiles, we report quantitative nucleotide analog interference mapping (QNAIM). This extension of the NAIM methodology uses the magnitude of analog interference as a function of ligand concentration to calculate binding constants for RNA with individual analog substitutions. In this way, QNAIM not only defines which functional groups are important to an interaction but simultaneously determines the energetic contribution made by each occurrence of that functional group within the RNA polymer. To establish the utility of this approach, QNAIM was used to quantify functional group interactions within the signal recognition particle (SRP), specifically the 4.5S RNA with the M domain of Ffh. In each of the cases in which energetic data were available from previous site-specific substitution analyses, QNAIM provided nearly equivalent results. These experiments on a model system demonstrate that QNAIM is an efficient method to establish a chemically detailed free energy profile for a wide variety of RNA-ligand interactions.     

Biochemical detection of monovalent metal ion binding sites within RNA

Many RNAs, including the ribosome, RNase P, and the group II intron, explicitly require monovalent cations for activity in vitro. Although the necessity of monovalent cations for RNA function has been known for more than a quarter of a century, the characterization of specific monovalent metal sites within large RNAs has been elusive. Here we describe a biochemical approach to identify functionally important monovalent cations in nucleic acids. This method uses thallium (Tl+), a soft Lewis acid heavy metal cation with chemical properties similar to those of the physiological alkaline earth metal potassium (K+). Nucleotide analog interference mapping (NAIM) with the sulfur-substituted nucleotide 6-thioguanosine in combination with selective metal rescue of the interference with Tl+ provides a distinct biochemical signature for monovalent metal ion binding. This approach has identified a K+ binding site within the P4-P6 domain of the Tetrahymena group I intron that is also present within the X-ray crystal structure. The technique also predicted a similar binding site within the Azoarcus group I intron where the structure is not known. The approach is applicable to any RNA molecule that can be transcribed in vitro and whose function can be assayed.     

Defining functional groups, core structural features and inter-domain tertiary contacts essential for group II intron self-splicing: a NAIM analysis.

Group II introns are self-splicing RNA molecules that are of considerable interest as ribozymes, mobile genetic elements and examples of folded RNA. Although these introns are among the most common ribozymes, little is known about the chemical and structural determinants for their reactivity. By using nucleotide analog interference mapping (NAIM), it has been possible to identify the nucleotide functional groups (Rp phosphoryls, 2'-hydroxyls, guanosine exocyclic amines, adenosine N7 and N6) that are most important for composing the catalytic core of the intron. The majority of interference effects occur in clusters located within the two catalytically essential Domains 1 and 5 (D1 and D5). Collectively, the NAIM results indicate that key tetraloop-receptor interactions display a specific chemical signature, that the epsilon-epsilon' interaction includes an elaborate array of additional features and that one of the most important core structures is an uncharacterized three-way junction in D1. By combining NAIM with site-directed mutagenesis, a new tertiary interaction, kappa-kappa', was identified between this region and the most catalytically important section of D5, adjacent to the AGC triad in stem 1. Together with the known zeta-zeta' interaction, kappa-kappa' anchors D5 firmly into the D1 scaffold, thereby presenting chemically essential D5 functionalities for participation in catalysis.     

NAIM and site-specific functional group modification analysis of RNase P RNA: magnesium dependent structure within the conserved P1-P4 multihelix junction contributes to catalysis

The tRNA processing endonuclease ribonuclease P contains an essential and highly conserved RNA molecule (RNase P RNA) that is the catalytic subunit of the enzyme. To identify and characterize functional groups involved in RNase P RNA catalysis, we applied self-cleaving ribozyme-substrate conjugates, on the basis of the RNase P RNA from Escherichia coli, in nucleotide analogue interference mapping (NAIM) and site-specific modification experiments. At high monovalent ion concentrations (3 M) that facilitate protein-independent substrate binding, we find that the ribozyme is largely insensitive to analogue substitution and that concentrations of Mg2+ (1.25 mM) well below that necessary for optimal catalytic rate (>100 mM) are required to produce interference effects because of modification of nucleotide bases. An examination of the pH dependence of the reaction rate at 1.25 mM Mg2+ indicates that the increased sensitivity to analogue interference is not due to a change in the rate-limiting step. The nucleotide positions detected by NAIM under these conditions are located exclusively in the catalytic domain, consistent with the proposed global structure of the ribozyme, and predominantly occur within the highly conserved P1-P4 multihelix junction. Several sensitive positions in J3/4 and J2/4 are proximal to a previously identified site of divalent metal ion binding in the P1-P4 element. Kinetic analysis of ribozymes with site-specific N7-deazaadenosine and deazaguanosine modifications in J3/4 was, in general, consistent with the interference results and also permitted the analysis of sites not accessible by NAIM. These results show that, in this region only, modification of the N7 positions of A62, A65, and A66 resulted in measurable effects on reaction rate and modification at each position displayed distinct sensitivities to Mg2+ concentration. These results reveal a restricted subset of individual functional groups within the catalytic domain that are particularly important for substrate cleavage and demonstrate a close association between catalytic function and metal ion-dependent structure in the highly conserved P1-P4 multihelix junction.     

Backbone and nucleobase contacts to glucosamine-6-phosphate in the glmS ribozyme

The glmS ribozyme resides in the 5' untranslated region of glmS mRNA and functions as a catalytic riboswitch that regulates amino sugar metabolism in certain Gram-positive bacteria. The ribozyme catalyzes self-cleavage of the mRNA and ultimately inhibits gene expression in response to binding of glucosamine-6-phosphate (GlcN6P), the metabolic product of the GlmS protein. We have used nucleotide analog interference mapping (NAIM) and suppression (NAIS) to investigate backbone and nucleobase functional groups essential for ligand-dependent ribozyme function. NAIM using GlcN6P as ligand identified requisite structural features and potential sites of ligand and/or metal ion interaction, whereas NAIS using glucosamine as ligand analog revealed those sites that orchestrate recognition of ligand phosphate. These studies demonstrate that the ligand-binding site lies in close proximity to the cleavage site in an emerging model of ribozyme structure that supports a role for ligand within the catalytic core.     

Ionization of a critical adenosine residue in the neurospora Varkud Satellite ribozyme active site

The Varkud Satellite (VS) ribozyme catalyzes a site-specific self-cleavage reaction that generates 5'-OH and 2',3'-cyclic phosphate products. Other ribozymes that perform an equivalent reaction appear to employ ionization of an active site residue, either to neutralize the negatively charged transition state or to act as a general acid-base catalyst. To test for important base ionization events in the VS ribozyme ligation reaction, we performed nucleotide analogue interference mapping (NAIM) with a series of ionization-sensitive adenosine and cytidine analogues. A756, a catalytically critical residue located within the VS active site, was the only nucleotide throughout the VS ribozyme that displayed the pH-dependent interference pattern characteristic of functional base ionization. We observed unique rescue of 8-azaadenosine (pK(a) 2.2) and purine riboside (pK(a) 2.1) interference at A756 at reduced reaction pH, suggestive of an ionization-specific effect. These results are consistent with protonation and/or deprotonation of A756 playing a direct role in the VS ribozyme reaction mechanism. In addition, NAIM experiments identified several functional groups within the RNA that play important roles in ribozyme folding and/or catalysis. These include residues in helix II, helix VI (730 loop), the II-III-VI and III-IV-V helix junctions, and loop V.     

Simulating RNA folding kinetics on approximated energy landscapes

We present a general computational approach to simulate RNA folding kinetics that can be used to extract population kinetics, folding rates and the formation of particular substructures that might be intermediates in the folding process. Simulating RNA folding kinetics can provide unique insight into RNA whose functions are dictated by folding kinetics and not always by nucleotide sequence or the structure of the lowest free-energy state. The method first builds an approximate map (or model) of the folding energy landscape from which the population kinetics are analyzed by solving the master equation on the map. We present results obtained using an analysis technique, map-based Monte Carlo simulation, which stochastically extracts folding pathways from the map. Our method compares favorably with other computational methods that begin with a comprehensive free-energy landscape, illustrating that the smaller, approximate map captures the major features of the complete energy landscape. As a result, our method scales to larger RNAs. For example, here we validate kinetics of RNA of more than 200 nucleotides. Our method accurately computes the kinetics-based functional rates of wild-type and mutant ColE1 RNAII and MS2 phage RNAs showing excellent agreement with experiment.     

RNA folding with soft constraints: reconciliation of probing data and thermodynamic secondary structure prediction

Thermodynamic folding algorithms and structure probing experiments are commonly used to determine the secondary structure of RNAs. Here we propose a formal framework to reconcile information from both prediction algorithms and probing experiments. The thermodynamic energy parameters are adjusted using 'pseudo-energies' to minimize the discrepancy between prediction and experiment. Our framework differs from related approaches that used pseudo-energies in several key aspects. (i) The energy model is only changed when necessary and no adjustments are made if prediction and experiment are consistent. (ii) Pseudo-energies remain biophysically interpretable and hold positional information where experiment and model disagree. (iii) The whole thermodynamic ensemble of structures is considered thus allowing to reconstruct mixtures of suboptimal structures from seemingly contradicting data. (iv) The noise of the energy model and the experimental data is explicitly modeled leading to an intuitive weighting factor through which the problem can be seen as folding with 'soft' constraints of different strength. We present an efficient algorithm to iteratively calculate pseudo-energies within this framework and demonstrate how this approach can be used in combination with SHAPE chemical probing data to improve secondary structure prediction. We further demonstrate that the pseudo-energies correlate with biophysical effects that are known to affect RNA folding such as chemical nucleotide modifications and protein binding.     

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.     

A folding control element for tertiary collapse of a group II intron ribozyme

Ribozymes derived from the group II intron ai5gamma collapse to a compact intermediate, folding to the native state through a slow, direct pathway that is unperturbed by kinetic traps. Molecular collapse of ribozyme D135 requires high magnesium concentrations and is thought to involve a structural element in domain 1 (D1). We used nucleotide analog interference mapping, in combination with nondenaturing gel electrophoresis, to identify RNA substructures and functional groups that are essential for D135 tertiary collapse. This revealed that the most crucial atoms for compaction are located within a small section of D1 that includes the kappa and zeta elements. This small substructure controls specific collapse of the molecule and, in later steps of the folding pathway, it forms the docking site for catalytic D5. In this way, the stage is set for proper active site formation during the earliest steps of ribozyme folding.     

Potential contact sites between the protein and RNA subunit in the Bacillus subtilis RNase P holoenzyme.

We have detected by nucleotide analog interference mapping (NAIM) AMPalphaS and IMPalphaS modifications in Bacillus subtilis RNase P RNA that interfere with binding of the homologous protein subunit. Interference as well as some enhancement effects were clustered in two main areas, in P10.1a/L10.1 and P12 of the specificity domain (cluster 1, domain I) and in P2, P3, P15.1, J18/2 and J19/4 of the catalytic domain (cluster 2a, domain II). Minor interferences in P1 and P19 and a strong and weak enhancement effect in P19 represent a third area located in domain II (cluster 2b). Our results suggest that P3, P2-J18/2 and J19/4 are key elements for anchoring of the protein to the catalytic domain close to the scissile phosphodiester in enzyme-substrate complexes. Sites of interference or enhancement in clusters 1 and 2a are located at distances between 65 and 130 A from each other in the current 3D model of a full-length RNase P RNA-substrate complex. Taking into account that the RNase P protein monomer can bridge a maximum distance of about 40 A, simultaneous direct contacts to the two aforementioned potential RNA-binding areas would be incompatible with our current understanding of bacterial RNase P RNA architecture. Our findings suggest that the current 3D model has to be rearranged in order to reduce the distance between clusters 1 and 2a. Alternatively, based on the recent finding that B. subtilis RNase P forms a tetramer consisting of two protein and two RNA subunits, cluster 1 may reflect one protein contact site in domain I, and cluster 2a a separate one in domain II.     

Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions

Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg²⁺ ion concentrations are low, K⁺ concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo–like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg²⁺ (0.5–2 mM) and K⁺ (140 mM) if the solution is supplemented with physiological amounts (∼20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperature-dependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution.     

Purine N7 groups that are crucial to the interaction of Escherichia coli rnase P RNA with tRNA.

We have detected by nucleotide analog interference mapping (NAIM) purine N7 functional groups in Escherichia coli RNase P RNA that are important for tRNA binding under moderate salt conditions (0.1 M Mg2+, 0.1 M NH4+). The majority of identified positions represent highly or universally conserved nucleotides. Our assay system allowed us, for the first time, to identify c7-deaza interference effects at two G residues (G292, G306). Several c7-deazaadenine interference effects (A62, A65, A136, A249, A334, A351) have also been identified in other studies performed at very different salt concentrations, either selecting for substrate binding in the presence of 0.025 M Ca2+ and 1 M NH4+ or self-cleavage of a ptRNA-RNase P RNA conjugate in the presence of 3 M NH4+ or Na+. This indicates that these N7 functional groups play a key role in the structural organization of ribozyme-substrate and -product complexes. We further observed that a c7-deaza modification at A76 of tRNA interferes with tRNA binding to and ptRNA processing by E. coli RNase P RNA. This finding combined with the strong c7-deaza interference at G292 of RNase P RNA supports a model in which substrate and product binding to E. coli RNase P RNA involves the formation of intermolecular base triples (A258-G292-C75 and G291-G259-A76).