RNA molecules with conserved catalytic cores but variable peripheries fold along unique energetically optimized pathways

Functional and kinetic constraints must be efficiently balanced during the folding process of all biopolymers. To understand how homologous RNA molecules with different global architectures fold into a common core structure we determined, under identical conditions, the folding mechanisms of three phylogenetically divergent group I intron ribozymes. These ribozymes share a conserved functional core defined by topologically equivalent tertiary motifs but differ in their primary sequence, size, and structural complexity. Time-resolved hydroxyl radical probing of the backbone solvent accessible surface and catalytic activity measurements integrated with structural-kinetic modeling reveal that each ribozyme adopts a unique strategy to attain the conserved functional fold. The folding rates are not dictated by the size or the overall structural complexity, but rather by the strength of the constituent tertiary motifs which, in turn, govern the structure, stability, and lifetime of the folding intermediates. A fundamental general principle of RNA folding emerges from this study: The dominant folding flux always proceeds through an optimally structured kinetic intermediate that has sufficient stability to act as a nucleating scaffold while retaining enough conformational freedom to avoid kinetic trapping. Our results also suggest a potential role of naturally selected peripheral A-minor interactions in balancing RNA structural stability with folding efficiency.     

Distinct sites of phosphorothioate substitution interfere with folding and splicing of the Anabaena group I intron

Although the active site of group I introns is phylogenetically conserved, subclasses of introns have evolved different mechanisms of stabilizing the catalytic core. Large introns contain weakly conserved 'peripheral' domains that buttress the core through predicted interhelical contacts, while smaller introns use loop-helix interactions for stability. In all cases, specific and non-specific magnesium ion binding accompanies folding into the active structure. Whether similar RNA-RNA and RNA-magnesium ion contacts play related functional roles in different introns is not clear, particularly since it can be difficult to distinguish interactions directly involved in catalysis from those important for RNA folding. Using phosphorothioate interference with RNA activity and structure in the small (249 nt) group I intron from Anabaena, we used two independent assays to detect backbone phosphates important for catalysis and those involved in intron folding. Comparison of the interference sites identified in each assay shows that positions affecting catalysis cluster primarily in the conserved core of the intron, consistent with conservation of functionally important phosphates, many of which are magnesium ion binding sites, in diverse group I introns, including those from Azoarcus and Tetrahymena. However, unique sites of folding interference located outside the catalytic core imply that different group I introns, even within the same subclass, use distinct sets of tertiary interactions to stabilize the structure of the catalytic core.     

Isolation and characterization of a family of stable RNA tetraloops with the motif YNMG that participate in tertiary interactions

RNA is known to fold into a variety of structural elements, many of which have sufficient sequence complexity to make the thermodynamic study of each possible variant impractical. We previously reported a method for isolating stable and unstable RNA sequences from combinatorial libraries using temperature gradient gel electrophoresis (TGGE). This method was used herein to analyze a six-nucleotide RNA hairpin loop library. Three rounds of in vitro selection were performed using TGGE, and unusually stable RNAs were identified by cloning and sequencing. Known stable tetraloops were found, including sequences belonging to the UNCG motif closed by a CG base pair, and the CUUG motif closed by a GC base pair. In addition, unknown tetraloops were found that were nearly as stable as cUNCGg, including sequences related through substitution of the U with a C (Y), the C with an A (M), or both. These substitutions allow hydrogen bonding and stacking interactions in the UNCG loop to be maintained. Thermodynamic analysis of YNMG and variant loops confirmed optimal stability with Y at position 1 and M at position 3. Similarity in structure and stability among YNMG loops was further supported by deoxyribose substitution, CD, and NMR experiments. A conserved tertiary interaction in 16S rRNA exists between a YAMG loop at position 343 and two adenines in the loop at position 159 (Escherichia coli numbering). NMR and functional group substitution experiments suggest that YNAG loops in particular have enhanced flexibility, which allows the tertiary interaction to be maintained with diverse loop sequences at position 159. Taken together, these results support the existence of an extended family of UNCG-like tetraloops with the motif cYNMGg that are thermodynamically stable and structurally similar and can engage in tertiary interactions in large RNA molecules.     

Comparative photocross-linking analysis of the tertiary structures of Escherichia coli and Bacillus subtilis RNase P RNAs.

Bacterial ribonuclease P contains a catalytic RNA subunit that cleaves precursor sequences from the 5' ends of pre-tRNAs. The RNase P RNAs from Bacillus subtilis and Escherichia coli each contain several unique secondary structural elements not present in the other. To understand better how these phylogenetically variable elements affect the global architecture of the ribozyme, photoaffinity cross-linking studies were carried out. Photolysis of photoagents attached at homologous sites in the two RNAs results in nearly identical cross-linking patterns, consistent with the homology of the RNAs and indicating that these RNAs contain a common, core tertiary structure. Distance constraints were used to derive tertiary structure models using a molecular mechanics-based modeling protocol. The resulting superimposition of large sets of equivalent models provides a low resolution (5-10 A) structure for each RNA. Comparison of these structure models shows that the conserved core helices occupy similar positions in space. Variably present helical elements that may play a role in global structural stability are found at the periphery of the core structure. The P5.1 and P15.1 helical elements, unique to the B.subtilis RNase P RNA, and the P6/16/17 helices, unique to the E.coli RNA, occupy similar positions in the structure models and, therefore, may have analogous structural function.     

Structural insights into the dynamics and function of the C-terminus of the E. coli RNA chaperone Hfq

The hexameric Escherichia coli RNA chaperone Hfq (Hfq(Ec)) is involved in riboregulation of target mRNAs by small trans-encoded RNAs. Hfq proteins of different bacteria comprise an evolutionarily conserved core, whereas the C-terminus is variable in length. Although the structure of the conserved core has been elucidated for several Hfq proteins, no structural information has yet been obtained for the C-terminus. Using bioinformatics, nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism (SRCD) spectroscopy and small angle X-ray scattering we provide for the first time insights into the conformation and dynamic properties of the C-terminal extension of Hfq(Ec). These studies indicate that the C-termini are flexible and extend laterally away from the hexameric core, displaying in this way features typical of intrinsically disordered proteins that facilitate intermolecular interactions. We identified a minimal, intrinsically disordered region of the C-terminus supporting the interactions with longer RNA fragments. This minimal region together with rest of the C-terminal extension provides a flexible moiety capable of tethering long and structurally diverse RNA molecules. Furthermore, SRCD spectroscopy supported the hypothesis that RNA fragments exceeding a certain length interact with the C-termini of Hfq(Ec).     

Studies on the structure and function of 16S ribosomal RNA using structure-specific chemical probes

Recent technological developments permit us to examine the accessibility of specific atoms on any nucleotide in any large RNA molecule to certain chemical probes. This can provide detailed information about the higher order structure of large RNA molecules, including secondary and tertiary structure, protein-RNA contacts, binding sites for functional ligands and possible biologically significant conformational changes. Here, we summarize recent studies on (i) the conformation of naked 16S rRNA under a variety of ionic conditions, and (ii) the behaviour of 16S rRNA in active and inactive 30S subunits, as defined by Zamir, Elson and their colleagues. The latter study reveals a reciprocal conformational change in the vicinity of the decoding region of 16S rRNA in 30S ribosomal subunits. This conformational change appears to be a rearrangement of tertiary and/or quaternary structure involving several universally conserved nucleotides. No reproducible effects are seen elsewhere in the molecule, suggesting that the active-inactive transition is a result of the observed conformational change.     

An algorithm for searching RNA motifs in genomic sequences

RNA molecules, which are found in all living cells, fold into characteristic structures that account for their diverse functional activities. Many of these RNA structures consist of a collection of fundamental RNA motifs. The various combinations of RNA basic components form different RNA classes and define their unique structural and functional properties. The availability of many genome sequences makes it possible to search computationally for functional RNAs. Biological experiments indicate that functional RNAs have characteristic RNA structural motifs represented by specific combinations of base pairings and conserved nucleotides in the loop regions. The searching for those well-ordered RNA structures and their homologues in genomic sequences is very helpful for the understanding of RNA-based gene regulation. In this paper, we consider the following problem: given an RNA sequence with a known secondary structure, efficiently determine candidate segments in genomic sequences that can potentially form RNA secondary structures similar to the given RNA secondary structure. Our new bottom-up approach searches all potential stem-loops similar to ones of the given RNA secondary structure first, and then based on located stem-loops, detects potential homologous structural RNAs in genomic sequences.     

RNAMotif, an RNA secondary structure definition and search algorithm

RNA molecules fold into characteristic secondary and tertiary structures that account for their diverse functional activities. Many of these RNA structures are assembled from a collection of RNA structural motifs. These basic building blocks are used repeatedly, and in various combinations, to form different RNA types and define their unique structural and functional properties. Identification of recurring RNA structural motifs will therefore enhance our understanding of RNA structure and help associate elements of RNA structure with functional and regulatory elements. Our goal was to develop a computer program that can describe an RNA structural element of any complexity and then search any nucleotide sequence database, including the complete prokaryotic and eukaryotic genomes, for these structural elements. Here we describe in detail a new computational motif search algorithm, RNAMotif, and demonstrate its utility with some motif search examples. RNAMotif differs from other motif search tools in two important aspects: first, the structure definition language is more flexible and can specify any type of base–base interaction; second, RNAMotif provides a user controlled scoring section that can be used to add capabilities that patterns alone cannot provide.     

Predicting the secondary structures and tertiary interactions of 211 group I introns in IE subgroup

The large number of currently available group I intron sequences in the public databases provides opportunity for studying this large family of structurally complex catalytic RNA by large-scale comparative sequence analysis. In this study, the detailed secondary structures of 211 group I introns in the IE subgroup were manually predicted. The secondary structure-favored alignments showed that IE introns contain 14 conserved stems. The P13 stem formed by long-range base-pairing between P2.1 and P9.1 is conserved among IE introns. Sequence variations in the conserved core divide IE introns into three distinct minor subgroups, namely IE1, IE2 and IE3. Co-variation of the peripheral structural motifs with core sequences supports that the peripheral elements function in assisting the core structure folding. Interestingly, host-specific structural motifs were found in IE2 introns inserted at S516 position. Competitive base-pairing is found to be conserved at the junctions of all long-range paired regions, suggesting a possible mechanism of establishing long-range base-pairing during large RNA folding. These findings extend our knowledge of IE introns, indicating that comparative analysis can be a very good complement for deepening our understanding of RNA structure and function in the genomic era.     

Structural basis of the enhanced stability of a mutant ribozyme domain and a detailed view of RNA--solvent interactions

BACKGROUND: The structure of P4-P6, a 160 nucleotide domain of the self-splicing Tetrahymena thermophila intron, was solved previously. Mutants of the P4-P6 RNA that form a more stable tertiary structure in solution were recently isolated by successive rounds of in vitro selection and amplification. RESULTS: We show that a single-site mutant (Delta C209) possessing greater tertiary stability than wild-type P4-P6 also crystallizes much more rapidly and under a wider variety of conditions. The crystal structure provides a satisfying explanation for the increased stability of the mutant; the deletion of C209 allows the adjacent bulged adenine to enter the P4 helix and form an A-G base pair, presumably attenuating the conformational flexibility of the helix. The structure of another mutant (Delta A210) was also solved and supports this interpretation. The crystals of Delta C209 diffract to a higher resolution limit than those of wild-type RNA (2.25 A versus 2.8 A), allowing assignment of innersphere and outersphere coordination contacts for 27 magnesium ions. Structural analysis reveals an intricate solvent scaffold with a preponderance of ordered water molecules on the inside rather than the surface of the folded RNA domain. CONCLUSIONS: In vitro evolution facilitated the identification of a highly stable, structurally homogeneous mutant RNA that was readily crystallizable. Analysis of the structure suggests that improving RNA secondary structure can stabilize tertiary structure and perhaps promote crystallization. In addition, the higher resolution model provides new details of metal ion-RNA interactions and identifies a core of ordered water molecules that may be integral to RNA tertiary structure formation.     

Discovery of 17 conserved structural RNAs in fungi

Many non-coding RNAs with known functions are structurally conserved: their intramolecular secondary and tertiary interactions are maintained across evolutionary time. Consequently, the presence of conserved structure in multiple sequence alignments can be used to identify candidate functional non-coding RNAs. Here, we present a bioinformatics method that couples iterative homology search with covariation analysis to assess whether a genomic region has evidence of conserved RNA structure. We used this method to examine all unannotated regions of five well-studied fungal genomes (Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Aspergillus fumigatus, and Schizosaccharomyces pombe). We identified 17 novel structurally conserved non-coding RNA candidates, which include four H/ACA box small nucleolar RNAs, four intergenic RNAs and nine RNA structures located within the introns and untranslated regions (UTRs) of mRNAs. For the two structures in the 3′ UTRs of the metabolic genes GLY1 and MET13, we performed experiments that provide evidence against them being eukaryotic riboswitches.     

Group II intron splicing in chloroplasts: identificationof mutations determining intron stability and fate of exon RNA.

In order to investigate in vivo splicing of group II introns in chloroplasts, we previously have integrated the mitochondrial intron rI1 from the green alga Scenedesmus obliquus into the Chlamydomonas chloroplast tscA gene. This construct allows a functional analysis of conserved intron sequences in vivo, since intron rI1 is correctly spliced in chloroplasts. Using site-directed mutagenesis, deletions of the conserved intron domains V and VI were performed. In another set of experiments, each possible substitution of the strictly conserved first intron nucleotide G1 was generated, as well as each possible single and double mutation of the tertiary base pairing gamma-gamma ' involved in the formation of the intron's tertiary RNA structure. In most cases, the intron mutations showed the same effect on in vivo intron splicing efficiency as they did on the in vitro self-splicing reaction, since catalytic activity is provided by the intron RNA itself. In vivo, all mutations have additional effects on the chimeric tscA -rI1 RNA, most probably due to the role played by trans -acting factors in intron processing. Substitutions of the gamma-gamma ' base pair lead to an accumulation of excised intron RNA, since intron stability is increased. In sharp contrast to autocatalytic splicing, all point mutations result in a complete loss of exon RNA, although the spliced intron accumulates to high levels. Intron degradation and exon ligation only occur in double mutants with restored base pairing between the gamma and gamma' sites. Therefore, we conclude that intron degradation, as well as the ligation of exon-exon molecules, depends on the tertiary intron structure. Furthermore, our data suggest that intron excision proceeds in vivo independent of ligation of exon-exon molecules.     

RNAlyzer—novel approach for quality analysis of RNA structural models

The continuously increasing amount of RNA sequence and experimentally determined 3D structure data drives the development of computational methods supporting exploration of these data. Contemporary functional analysis of RNA molecules, such as ribozymes or riboswitches, covers various issues, among which tertiary structure modeling becomes more and more important. A growing number of tools to model and predict RNA structure calls for an evaluation of these tools and the quality of outcomes their produce. Thus, the development of reliable methods designed to meet this need is relevant in the context of RNA tertiary structure analysis and can highly influence the quality and usefulness of RNA tertiary structure prediction in the nearest future. Here, we present RNAlyzer—a computational method for comparison of RNA 3D models with the reference structure and for discrimination between the correct and incorrect models. Our approach is based on the idea of local neighborhood, defined as a set of atoms included in the sphere centered around a user-defined atom. A unique feature of the RNAlyzer is the simultaneous visualization of the model-reference structure distance at different levels of detail, from the individual residues to the entire molecules.     

Coupled nucleotide covariations reveal dynamic RNA interaction patterns

Evolutionarily conserved structures in related RNA molecules contain coordinated variations (covariations) of paired nucleotides. Analysis of covariations is a very powerful approach to deduce phylogenetically conserved (i.e., functional) conformations, including tertiary interactions. Here we discuss conserved RNA folding pathways that are revealed by covariation patterns. In such pathways, structural requirements for alternative pairings cause some nucleotides to covary with two different partners. Such "coupled" covariations between three or more nucleotides were found in various types of RNAs. The analysis of coupled covariations can unravel important features of RNA folding dynamics and improve phylogeny reconstruction in some cases. Importantly, it is necessary to distinguish between multiple covariations determined by mutually exclusive structures and those determined by tertiary contacts.     

Thiophosphate interference experiments locate phosphates important for the hammerhead RNA self-cleavage reaction.

A hammerhead domain of less than 50 nucleotides is responsible for a self-cleavage reaction in the replication of plant RNA pathogens. The hammerhead is composed of three helices joining at a central conserved core of 11 single stranded nucleotides. The core is believed to fold into a tertiary structure that provides functional groups for catalysis and to coordinate one or more divalent metal ions. In this study we use a phosphorothioate substitution interference assay to identify four phosphates in the conserved core which also play a role in the self-cleavage reaction.     

Detailed analysis of RNA-protein interactions within the ribosomal protein S8-rRNA complex from the archaeon Methanococcus jannaschii

The crystal structure of ribosomal protein S8 bound to its target 16 S rRNA from a hyperthermophilic archaeon Methanococcus jannaschii has been determined at 2.6 A resolution. The protein interacts with the minor groove of helix H21 at two sites located one helical turn apart, with S8 forming a bridge over the RNA major groove. The specificity of binding is essentially provided by the C-terminal domain of S8 and the highly conserved nucleotide core, characterized by two dinucleotide platforms, facing each other. The first platform (A595-A596), which is the less phylogenetically and structurally constrained, does not directly contact the protein but has an important shaping role in inducing cross-strand stacking interactions. The second platform (U641-A642) is specifically recognized by the protein. The universally conserved A642 plays a pivotal role by ensuring the cohesion of the complex organization of the core through an array of hydrogen bonds, including the G597-C643-U641 base triple. In addition, A642 provides the unique base-specific interaction with the conserved Ser105, while the Thr106 - Thr107 peptide link is stacked on its purine ring. Noteworthy, the specific recognition of this tripeptide (Thr-Ser-Thr/Ser) is parallel to the recognition of an RNA tetraloop by a dinucleotide platform in the P4-P6 ribozyme domain of group I intron. This suggests a general dual role of dinucleotide platforms in recognition of RNA or peptide motifs. One prominent feature is that conserved side-chain amino acids, as well as conserved bases, are essentially involved in maintaining tertiary folds. The specificity of binding is mainly driven by shape complementarity, which is increased by the hydrophobic part of side-chains. The remarkable similarity of this complex with its homologue in the T. thermophilus 30 S subunit indicates a conserved interaction mode between Archaea and Bacteria.     

Structure-function analysis from the outside in: long-range tertiary contacts in RNA exhibit distinct catalytic roles

The conserved catalytic core of the Tetrahymena group I ribozyme is encircled by peripheral elements. We have conducted a detailed structure-function study of the five long-range tertiary contacts that fasten these distal elements together. Mutational ablation of each of the tertiary contacts destabilizes the folded ribozyme, indicating a role of the peripheral elements in overall stability. Once folded, three of the five tertiary contact mutants exhibit defects in overall catalysis that range from 20- to 100-fold. These and the subsequent results indicate that the structural ring of peripheral elements does not act as a unitary element; rather, individual connections have distinct roles as further revealed by kinetic and thermodynamic dissection of the individual reaction steps. Ablation of P14 or the metal ion core/metal ion core receptor (MC/MCR) destabilizes docking of the substrate-containing P1 helix into tertiary interactions with the ribozyme's conserved core. In contrast, ablation of the L9/P5 contact weakens binding of the guanosine nucleophile by slowing its association, without affecting P1 docking. The P13 and tetraloop/tetraloop receptor (TL/TLR) mutations had little functional effect and small, local structural changes, as revealed by hydroxyl radical footprinting, whereas the P14, MC/MCR, and L9/P5 mutants show structural changes distal from the mutation site. These changes extended into regions of the catalytic core involved in docking or guanosine binding. Thus, distinct allosteric pathways couple the long-range tertiary contacts to functional sites within the conserved core. This modular functional specialization may represent a fundamental strategy in RNA structure-function interrelationships.