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MTR1 is an in vitro-selected alkyl transferase ribozyme that transfers an alkyl group from O 6 -alkylguanine to N1 of the target adenine in the RNA substrate (A63). The structure of the ribozyme suggested a mechanism in which a cytosine (C10) acts as a general acid to protonate O 6 -alkylguanine N1. Here, we have analyzed the role of the C10 general acid and the A63 nucleophile by atomic mutagenesis and computation. C10 was substituted by n1c and n1c, c5n variants. The n1c variant has an elevated p K a (11.4 as the free nucleotide) and leads to a 10 4 -fold lower activity that is pH-independent. Addition of the second c5n substitution with a lower p K a restored both the rate and pH dependence of alkyl transfer. Quantum mechanical calculations indicate that protonation of O 6 -alkylguanine lowers the barrier to alkyl transfer and that there is a significantly elevated barrier to proton transfer for the n1c single substitution. The calculated p K a values are in good agreement with the apparent values from measured rates. Increasing the p K a of the nucleophile by A63 n7c substitution led to a 6-fold higher rate. The increased reactivity of the nucleophile corresponds to a β nuc of ∼0.5, indicating significant C-N bond formation in the transition state. Taken together, these results are consistent with a two-step mechanism comprising protonation of the O 6 -alkylguanine followed by alkyl transfer., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

We have determined the crystal structure of a pseudoknot (PK)-containing hammerhead ribozyme that closely resembles the pistol ribozyme, with essentially identical secondary structure and connectivity. The activity is more sensitive to deletion of the G8 2'OH than to the absence of magnesium ions, indicating that the catalytic mechanism is the same as the extended hammerhead, and distinct from the pistol ribozyme. Here we show that nucleophilic attack is almost perfectly in-line, and the G8 2'OH is well positioned to act as general acid, being directed towards the O5' leaving group, and 2.9 Å away from it. Despite the similarity in overall structure to the pistol ribozyme, the local structure close to the cleavage site differs, and the PK hammerhead retains its unique mechanistic identity and demonstrates enhanced activity over other hammerhead ribozymes under standard conditions., (© 2024. The Author(s).)

Riboswitches are regulatory elements found in bacterial mRNAs that control downstream gene expression through ligand-induced conformational changes. Here, we used single-molecule FRET to map the conformational landscape of the translational SAM/SAH riboswitch and probe how co-transcriptional ligand-induced conformational changes affect its translation regulation function. Riboswitch folding is highly heterogeneous, suggesting a rugged conformational landscape that allows for sampling of the ligand-bound conformation even in the absence of ligand. The addition of ligand shifts the landscape, favoring the ligand-bound conformation. Mutation studies identified a key structural element, the pseudoknot helix, that is crucial for determining ligand-free conformations and their ligand responsiveness. We also investigated ribosomal binding site accessibility under two scenarios: pre-folding and co-transcriptional folding. The regulatory function of the SAM/SAH riboswitch involves kinetically favoring ligand binding, but co-transcriptional folding reduces this preference with a less compact initial conformation that exposes the Shine-Dalgarno sequence and takes min to redistribute to more compact conformations of the pre-folded riboswitch. Such slow equilibration decreases the effective ligand affinity. Overall, our study provides a deeper understanding of the complex folding process and how the riboswitch adapts its folding pattern in response to ligand, modulates ribosome accessibility and the role of co-transcriptional folding in these processes., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

k-Junctions are elaborated forms of kink turns with an additional helix on the nonbulged strand, thus forming a three-way helical junction. Two were originally identified in the structures of Arabidopsis and Escherichia coli thiamine pyrophosphate (TPP) riboswitches, and another called DUF-3268 was tentatively identified from sequence information. In this work we show that the Arabidopsis and E. coli riboswitch k-junctions fold in response to the addition of magnesium or sodium ions, and that atomic mutations that should disrupt key hydrogen bonding interactions greatly impair folding. Using X-ray crystallography, we have determined the structure of the DUF-3268 RNA and thus confirmed that it is a k-junction. It also folds upon the addition of metal ions, though requiring a 40-fold lower concentration of either divalent or monovalent ions. The key difference between the DUF-3268 and riboswitch k-junctions is the lack of nucleotides inserted between G1b and A2b in the former. We show that this insertion is primarily responsible for the difference in folding properties. Finally, we show that the DUF-3268 can functionally substitute for the k-junction in the E. coli TPP riboswitch such that the chimera can bind the TPP ligand, although less avidly., (© 2023 Li et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

ANKLE1 is a nuclease that provides a final opportunity to process unresolved junctions in DNA that would otherwise create chromosomal linkages blocking cell division. It is a GIY-YIG nuclease. We have expressed an active domain of human ANKLE1 containing the GIY-YIG nuclease domain in bacteria, that is monomeric in solution and when bound to a DNA Y-junction, and unilaterally cleaves a cruciform junction. Using an AlphaFold model of the enzyme we identify the key active residues, and show that mutation of each leads to impairment of activity. There are two components in the catalytic mechanism. Cleavage rate is pH dependent, corresponding to a pKa of 6.9, suggesting an involvement of the conserved histidine in proton transfer. The reaction rate depends on the nature of the divalent cation, likely bound by glutamate and asparagine side chains, and is log-linear with the metal ion pKa. We propose that the reaction is subject to general acid-base catalysis, using a combination of tyrosine and histidine acting as general base and water directly coordinated to the metal ion as general acid. The reaction is temperature dependent; activation energy Ea = 37 kcal mol-1, suggesting that cleavage is coupled to opening of DNA in the transition state., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A methyltransferase ribozyme (MTR1) was selected in vitro to catalyze alkyl transfer from exogenous O6-methylguanine (O6mG) to a target adenine N1, and recently, high-resolution crystal structures have become available. We use a combination of classical molecular dynamics, ab initio quantum mechanical/molecular mechanical (QM/MM) and alchemical free energy (AFE) simulations to elucidate the atomic-level solution mechanism of MTR1. Simulations identify an active reactant state involving protonation of C10 that hydrogen bonds with O6mG:N1. The deduced mechanism involves a stepwise mechanism with two transition states corresponding to proton transfer from C10:N3 to O6mG:N1 and rate-controlling methyl transfer (19.4  kcal·mol-1 barrier). AFE simulations predict the pKa for C10 to be 6.3, close to the experimental apparent pKa of 6.2, further implicating it as a critical general acid. The intrinsic rate derived from QM/MM simulations, together with pKa calculations, enables us to predict an activity-pH profile that agrees well with experiment. The insights gained provide further support for a putative RNA world and establish new design principles for RNA-based biochemical tools., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We present crystal structures of a new NAD+-binding riboswitch termed NAD+-II, bound to nicotinamide mononucleotide (NMN), nicotinamide adenine dinucleotide (NAD+) and nicotinamide riboside (NR). The RNA structure comprises a number of structural features including three helices, one of which forms a triple helix by interacting with an A5 strand in its minor-groove, and another formed from a long-range pseudoknot. The core of the structure (centrally located and coaxial with the triplex and the pseudoknot) includes two consecutive quadruple base interactions. Unusually the riboswitch binds two molecules of ligand, bound at distinct, non-overlapping sites in the RNA. Binding occurs primarily through the nicotinamide moiety of each ligand, held by specific hydrogen bonding and stacking interactions with the pyridyl ring. The mode of binding is the same for NMN, NR and the nicotinamide moiety of NAD+. In addition, when NAD+ is bound into one site it adopts an elongated conformation such that its diphosphate linker occupies a groove on the surface of the RNA, following which the adenine portion inserts into a pocket and makes specific hydrogen bonding interactions. Thus the NAD+-II riboswitch is distinct from the NAD+-I riboswitch in that it binds two molecules of ligand at separate sites, and that binding occurs principally through the nicotinamide moiety., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Ribozymes are excellent systems in which to study 'sequence - structure - function' relationships in RNA molecules. Understanding these relationships may greatly help structural modeling and design of functional RNA structures and some functional structural modules could be repurposed in molecular design. At present, there is no comprehensive database summarising all the natural ribozyme families. We have therefore created Ribocentre, a database that collects together sequence, structure and mechanistic data on 21 ribozyme families. This includes available information on timelines, sequence families, secondary and tertiary structures, catalytic mechanisms, applications of the ribozymes together with key publications. The database is publicly available at https://www.ribocentre.org., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Resolution of Holliday junctions is a critical intermediate step of homologous recombination in which junctions are processed by junction-resolving endonucleases. Although binding and cleavage are well understood, the question remains how the enzymes locate their substrate within long duplex DNA. Here we track fluorescent dimers of endonuclease I on DNA, presenting the complete single-molecule reaction trajectory for a junction-resolving enzyme finding and cleaving a Holliday junction. We show that the enzyme binds remotely to dsDNA and then undergoes 1D diffusion. Upon encountering a four-way junction, a catalytically-impaired mutant remains bound at that point. An active enzyme, however, cleaves the junction after a few seconds. Quantitative analysis provides a comprehensive description of the facilitated diffusion mechanism. We show that the eukaryotic junction-resolving enzyme GEN1 also undergoes facilitated diffusion on dsDNA until it becomes located at a junction, so that the general resolution trajectory is probably applicable to many junction resolving enzymes., (© 2022. The Author(s).)

Known ribozymes in contemporary biology perform a limited range of chemical catalysis, but in vitro selection has generated species that catalyze a broader range of chemistry; yet, there have been few structural and mechanistic studies of selected ribozymes. A ribozyme has recently been selected that can catalyze a site-specific methyl transfer reaction. We have solved the crystal structure of this ribozyme at a resolution of 2.3 Å, showing how the RNA folds to generate a very specific binding site for the methyl donor substrate. The structure immediately suggests a catalytic mechanism involving a combination of proximity and orientation and nucleobase-mediated general acid catalysis. The mechanism is supported by the pH dependence of the rate of catalysis. A selected methyltransferase ribozyme can thus use a relatively sophisticated catalytic mechanism, broadening the range of known RNA-catalyzed chemistry., (© 2022. The Author(s).)

It is commonly thought that in the early development of life on this planet RNA would have acted both as a store of genetic information and as a catalyst. While a number of RNA enzymes are known in contemporary cells, they are largely confined to phosphoryl transfer reactions, whereas an RNA based metabolism would have required a much greater chemical diversity of catalysis. Here we discuss how RNA might catalyze a wider variety of chemistries, and particularly how information gleaned from riboswitches could suggest how ribozymes might recruit coenzymes to expand their chemical range. We ask how we might seek such activities in modern biology. This article is categorized under: RNA-Based Catalysis > Miscellaneous RNA-Catalyzed Reactions Regulatory RNAs/RNAi/Riboswitches > Riboswitches RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry., (© 2021 The Authors. WIREs RNA published by Wiley Periodicals LLC.)

k-Turns are widespread key architectural elements that occur in many classes of RNA molecules. We have shown previously that their folding properties (whether or not they fold into their tightly kinked structure on addition of metal ions) and conformation depend on their local sequence, and we have elucidated a series of rules for prediction of these properties from sequence. In this work, we have expanded the rules for prediction of folding properties, and then applied the full set to predict the folding and conformation of four probable k-turns we have identified amongst 224 structured RNA species found in bacterial intergenenic regions by the Breaker lab (1). We have analyzed the ion-dependence of folding of the four k-turns using fluorescence resonance energy transfer, and determined the conformation of two of them using X-ray crystallography. We find that the experimental data fully conform to both the predicted folding and conformational properties. We conclude that our folding rules are robust, and can be applied to new k-turns of unknown characteristics with confidence., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

All physical connections between sister chromatids must be broken before cells can divide, and eukaryotic cells have evolved multiple ways in which to process branchpoints connecting DNA molecules separated both spatially and temporally. A single DNA link between chromatids has the potential to disrupt cell cycle progression and genome integrity, so it is highly likely that cells require a nuclease that can process remaining unresolved and hemi-resolved DNA junctions and other branched species at the very late stages of mitosis. We argue that ANKLE1 probably serves this function in human cells (LEM-3 in Caenorhabditis elegans). LEM-3 has previously been shown to be located at the cell mid-body, and we show here that human ANKLE1 is a nuclease that cleaves a range of branched DNA species. It thus has the substrate selectivity consistent with an enzyme required to process a variety of unresolved and hemi-resolved branchpoints in DNA. Our results suggest that ANKLE1 acts as a catch-all enzyme of last resort that allows faithful chromosome segregation and cell division to occur., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

While most SAM riboswitches strongly discriminate between SAM and SAH, the SAM/SAH riboswitch responds to both ligands with similar apparent affinities. We have determined crystal structures of the SAM/SAH riboswitch bound to SAH, SAM and other variant ligands at high resolution. The riboswitch forms an H-type pseudoknot structure with coaxial alignment of the stem-loop helix (P1) and the pseudoknot helix (PK). An additional three base pairs form at the non-open end of P1, and the ligand is bound at the interface between the P1 extension and the PK helix. The adenine nucleobase is stacked into the helix and forms a trans Hoogsteen-Watson-Crick base pair with a uridine, thus becoming an integral part of the helical structure. The majority of the specific interactions are formed with the adenosine. The methionine or homocysteine chain lies in the groove making a single hydrogen bond, and there is no discrimination between the sulfonium of SAM or the thioether of SAH. Single-molecule FRET analysis reveals that the riboswitch exists in two distinct conformations, and that addition of SAM or SAH shifts the population into a stable state that likely corresponds to the form observed in the crystal. A model for translational regulation is presented whereby in the absence of ligand the riboswitch is largely unfolded, lacking the PK helix so that translation can be initiated at the ribosome binding site. But the presence of ligand stabilizes the folded conformation that includes the PK helix, so occluding the ribosome binding site and thus preventing the initiation of translation., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The nad A motif is the first known NAD + -dependent riboswitch, comprising two similar tandem bulged stem-loop structures. We have determined the structure of the 5' domain 1 of the riboswitch. It has three coaxial helical segments, separated by an ACANCCCC bulge and by an internal loop, with a tertiary contact between them that includes two C:G base pairs. We have determined the structure with a number of ligands related to NADH, but in each case only the ADP moiety is observed. The adenosine adopts an anti conformation, forms multiple hydrogen bonds across the width of the sugar edge of the penultimate C:G base pair of the helix preceding the bulge, and the observed contacts have been confirmed by mutagenesis and calorimetry. Two divalent metal ions play a key structural role at the narrow neck of the bulge. One makes direct bonding contacts to the diphosphate moiety, locking it into position. Thus the nucleobase, ribose, and phosphate groups of the ADP moiety are all specifically recognized by the RNA. The NAD + riboswitch is modular. Domain 1 is an ADP binding domain that may be ancient and could potentially be used in combination with other ligand binding motifs such as CoA., (© 2020 Huang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Box C/D RNA protein complexes (RNPs) catalyze site-specific 2'-O-methylation of RNA with specificity determined by guide RNAs. In eukaryotic C/D RNP, the paralogous Nop58 and Nop56 proteins specifically associate with terminal C/D and internal C'/D' motifs of guide RNAs, respectively. We have reconstituted active C/D RNPs with recombinant proteins of the thermophilic yeast Chaetomium thermophilum. Nop58 and Nop56 could not distinguish between the two C/D motifs in the reconstituted enzyme, suggesting that the assembly specificity is imposed by trans-acting factors in vivo. The two C/D motifs are functionally independent and halfmer C/D RNAs can also guide site-specific methylation. Extensive pairing between C/D RNA and substrate is inhibitory to modification for both yeast and archaeal C/D RNPs. N6-methylated adenine at box D/D' interferes with the function of the coupled guide. Our data show that all C/D RNPs share the same functional organization and mechanism of action and provide insight into the assembly specificity of eukaryotic C/D RNPs., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

N 6 -methyladenine is the most common covalent modification in cellular RNA species, with demonstrated functional consequences. At the molecular level this methylation could alter local RNA structure, and/or modulate the binding of specific proteins. We have previously shown that trans -Hoogsteen-sugar (sheared) A:G base pairs can be completely disrupted by methylation, and that this occurs in a sub-set ofD/D k-turn structures. In this work we have investigated to what extent sequence context affects the severity with which inclusion of N 6 -methyladenine into different A:G base pairs of a standard k-turn affects RNA folding and L7Ae protein binding. We find that local sequence has a major influence, ranging from complete absence of folding and protein binding to a relatively mild effect. We have determined the crystal structure of one of these species both free and protein-bound, showing the environment of the methyl group and the way the modification is accommodated into the k-turn structure.

We have determined the structure of the glutamine-II riboswitch ligand binding domain using X-ray crystallography. The structure was solved using a novel combination of homology modeling and molecular replacement. The structure comprises three coaxial helical domains, the central one of which is a pseudoknot with partial triplex character. The major groove of this helix provides the binding site for L-glutamine, which is extensively hydrogen bonded to the RNA. Atomic mutation of the RNA at the ligand binding site leads to loss of binding shown by isothermal titration calorimetry, explaining the specificity of the riboswitch. A metal ion also plays an important role in ligand binding. This is directly bonded to a glutamine carboxylate oxygen atom, and its remaining inner-sphere water molecules make hydrogen bonding interactions with the RNA., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The nucleolytic ribozymes carry out site-specific RNA cleavage reactions by nucleophilic attack of the 2'-oxygen atom on the adjacent phosphorus with an acceleration of a million-fold or greater. A major part of this arises from concerted general acid-base catalysis. Recent identification of new ribozymes has expanded the group to a total of nine and this provides a new opportunity to identify sub-groupings according to the nature of the general base and acid. These include nucleobases, hydrated metal ions, and 2'-hydroxyl groups. Evolution has selected a number of different combinations of these elements that lead to efficient catalysis. These differences provide a new mechanistic basis for classifying these ribozymes., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

Comparison of the secondary and three-dimensional structures of the hammerhead and pistol ribozymes reveals many close similarities, so in this work we have asked if they are mechanistically identical. We have determined a new crystal structure of the pistol ribozyme and have shown that G40 acts as general base in the cleavage reaction. The conformation in the active site ensures an in-line attack of the O2' nucleophile, and the conformation at the scissile phosphate and the position of the general base are closely similar to those in the hammerhead ribozyme. However, the two ribozymes differ in the nature of the general acid. 2'-Amino substitution experiments indicate that the general acid of the hammerhead ribozyme is the O2' of G8, while that of the pistol ribozyme is a hydrated metal ion. The two ribozymes are related but mechanistically distinct.

We have designed structure-based ligands for the guanidine-II riboswitch that bind with enhanced affinity, exploiting the twin binding sites created by loop-loop interaction. We synthesized diguanidine species, comprising two guanidino groups covalently connected by C n linkers where n = 4 or 5. Calorimetric and fluorescent analysis shows that these ligands bind with a 10-fold higher affinity to the riboswitch compared to guanidine. We determined X-ray crystal structures of the riboswitch bound to the new ligands, showing that the guanidino groups are bound to both nucleobases and backbone within the binding pockets, analogously to guanidine binding. The connecting chain passes through side openings in the binding pocket and traverses the minor groove of the RNA. The combination of the riboswitch loop-loop interaction and our novel ligands has potential applications in chemical biology., (© 2019 Huang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Holliday junction (HJ) resolution by resolving enzymes is essential for chromosome segregation and recombination-mediated DNA repair. HJs undergo two types of structural dynamics that determine the outcome of recombination: conformer exchange between two isoforms and branch migration. However, it is unknown how the preferred branch point and conformer are achieved between enzyme binding and HJ resolution given the extensive binding interactions seen in static crystal structures. Single-molecule fluorescence resonance energy transfer analysis of resolving enzymes from bacteriophages (T7 endonuclease I), bacteria (RuvC), fungi (GEN1) and humans (hMus81-Eme1) showed that both types of HJ dynamics still occur after enzyme binding. These dimeric enzymes use their multivalent interactions to achieve this, going through a partially dissociated intermediate in which the HJ undergoes nearly unencumbered dynamics. This evolutionarily conserved property of HJ resolving enzymes provides previously unappreciated insight on how junction resolution, conformer exchange and branch migration may be coordinated.

A recent study has shown that archaeal L7Ae binds to a putative k-turn structure in the 5'-leader of the mRNA of its structural gene to regulate translation. To function as a regulator, the RNA should be unstructured in the absence of protein, but it should adopt a k-turn-containing stem-loop on binding L7Ae. Sequence analysis of UTR sequences indicates that their k-turn elements will be unable to fold in the absence of L7Ae, and we have demonstrated this experimentally in solution using FRET for the Archaeoglobus fulgidus sequence. We have solved the X-ray crystal structure of the complex of the A. fulgidus RNA bound to its cognate L7Ae protein. The RNA adopts a standard k-turn conformation that is specifically recognized by the L7Ae protein, so stabilizing the stem-loop. In-line probing of the natural-sequence UTR shows that the RNA is unstructured in the absence of L7Ae binding, but folds on binding the protein such that the ribosome binding site is occluded. Thus, L7Ae regulates its own translation by switching the conformation of the RNA to alter accessibility., (© 2019 Huang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

GEN1 is a member of the FEN/EXO family of structure-selective nucleases that cleave 1 nt 3' to a variety of branchpoints. For each, the H2TH motif binds a monovalent ion and plays an important role in binding one helical arm of the substrates. We investigate here the importance of this metal ion on substrate specificity and GEN1 structure. In the presence of K+ ions the substrate specificity is wider than in Na+, yet four-way junctions remain the preferred substrate. In a combination of K+ and Mg2+ second strand cleavage is accelerated 17-fold, ensuring bilateral cleavage of the junction. We have solved crystal structures of Chaetomium thermophilum GEN1 with Cs+, K+ and Na+ bound. With bound Cs+ the loop of the H2TH motif extends toward the active site so that D199 coordinates a Mg2+, buttressed by an interaction of the adjacent Y200. With the lighter ions bound the H2TH loop changes conformation and retracts away from the active site. We hypothesize this conformational change might play a role in second strand cleavage acceleration.

SAM-V is one of the class of riboswitches that bind S-adenosylmethione, regulating gene expression by controlling translation. We have solved the crystal structure of the metY SAM-V riboswitch bound to its SAM ligand at 2.5 Å resolution. The RNA folds as an H-type pseudoknot, with a major-groove triple helix in which resides the SAM ligand binding site. The bound SAM adopts an elongated conformation aligned with the axis of the triple helix, and is held at either end by hydrogen bonding to the adenine and the amino acid moieties. The central sulfonium cation makes electrostatic interactions with an U:A.U base triple, so conferring specificity. We propose a model in which SAM binding leads to association of the triplex third strand that stabilizes a short helix and occludes the ribosome binding site. Thus the new structure explains both ligand specificity and the mechanism of genetic control.

Four-way Holliday junctions in DNA are the central intermediates of genetic recombination and must be processed into regular duplex species. One mechanism for achieving this is called resolution, brought about by structure-selective nucleases. GEN1 is an important junction-resolving enzyme in eukaryotic cells, a member of the FEN1/EXO1 superfamily of nucleases. While human GEN1 is difficult to work with because of aggregation, orthologs from thermophilic fungi have been identified using bioinformatics and have proved to have excellent properties. Here, the expression and purification of this enzyme from Chaetomium thermophilum is described, together with the means of investigating its biochemical properties. The enzyme is quite similar to junction-resolving enzymes from lower organisms, binding to junctions in dimeric form, introducing symmetrical bilateral cleavages, the second of which is accelerated to promote productive resolution. Crystallization of C. thermophilum GEN1 is described, and the structure of a DNA-product complex. Juxtaposition of complexes in the crystal lattice suggests how the structure of a dimeric enzyme with an intact junction is organized., (© 2018 Elsevier Inc. All rights reserved.)

The kink-turn (k-turn) is a widespread structural motif found in functional RNA species. It typically comprises a three-nucleotide bulge followed by tandem trans sugar edge-Hoogsteen G:A base pairs. It introduces a sharp kink into the axis of duplex RNA, juxtaposing the minor grooves. Cross-strand H-bonds form at the interface, accepted by the conserved adenine nucleobases of the G:A basepairs. Alternative acceptors for one of these divides the k-turns into two conformational classes N3 and N1. The base pair that follows the G:A pairs (3b:3n) determines which conformation is adopted by a given k-turn. k-turns often mediate tertiary contacts in folded RNA species and frequently bind proteins. Common k-turn binding proteins include members of the L7Ae family, such as the human 15·5k protein. A recognition helix within these proteins binds in the widened major groove on the outside of the k-turn, that makes specific H-bonds with the conserved guanine nucleobases of the G:A pairs. L7Ae binds with extremely high affinity, and single-molecule data are consistent with folding by conformational selection. The standard, simple k-turn can be elaborated in a variety of ways, that include the complex k-turns and the k-junctions. In free solution in the absence of added metal ions or protein k-turns do not adopt the tightly-kinked conformation. They undergo folding by the binding of proteins, by the formation of tertiary contacts, and some (but not all) will fold on the addition of metal ions. Whether or not folding occurs in the presence of metal ions depends on local sequence, including the 3b:3n position, and the -1b:-1n position (5' to the bulge). In most cases -1b:-1n = C:G, so that the 3b:3n position is critical since it determines both folding properties and conformation. In general, the selection of these sequence matches a given k-turn to its biological requirements. The k-turn structure is now very well understood, to the point at which they can be used as a building block for the formation of RNA nano-objects, including triangles and squares.

The indodicarbocyanine fluorophores Cy3 and Cy5 are extensively used as donor-acceptor pairs in fluorescence resonance energy transfer experiments, especially those involving single molecules. When terminally attached to double-stranded nucleic acids via the 5' phosphate group these fluorophores stack onto the ends of the molecule. Knowledge of the positions of the fluorophores is critical to the interpretation of fluorescence resonance energy transfer data. The positions have been demonstrated for double-stranded (ds) DNA using NMR spectroscopy. Here, we have used x-ray crystallography to analyze the location of Cy3 and Cy5 on dsRNA, using complexes of an RNA stem-loop bound to L5 protein determined at 2.4 Å resolution. This confirms the tendency of both fluorophores to stack on the free end of RNA, with the long axis of the fluorophores approximately parallel to that of the terminal basepair. However, the manner of interaction of both Cy3 and Cy5 with the terminus of the dsRNA is significantly different from that deduced for dsDNA using NMR. The fluorophores are stacked on the terminal basepair such that their indole nitrogen atoms lie on the major groove side, and thus their pendant methyl groups are on the minor groove side., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Folding properties differ markedly between kink-turns (k-turns) that have different biological functions. While ribosomal and riboswitch k-turns generally fold into their kinked conformation on addition of metal ions, box C/D snoRNP k-turns remain completely unfolded under these conditions, although they fold on addition of L7Ae protein. Sequence elements have been systematically exchanged between a standard ribosomal k-turn (Kt-7) that folds on addition of metal ions, and a box C/D k-turn. Folding was studied using fluorescence resonance energy transfer and gel electrophoresis. Three sequence elements each contribute in an approximately additive manner to the different folding properties of Kt-7 and box C/D k-turns from archaea. Bioinformatic analysis indicates that k-turn sequences evolve sequences that suit their folding properties to their biological function. The majority of ribosomal and riboswitch k-turns have sequences allowing unassisted folding in response to the presence of metal ions. In contrast, box C/D k-turns have sequences that require the binding of proteins to drive folding into the kinked conformation, consistent with their role in the assembly of the box C/D snoRNP apparatus. The rules governing the influence of sequence on folding properties can be applied to other standard k-turns to predict their folding characteristics., (© 2017 Ashraf et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Riboswitches are structural elements found in mRNA molecules that couple small-molecule binding to regulation of gene expression, usually by controlling transcription or translation. We have determined high-resolution crystal structures of the ykkC guanidine III riboswitch from Thermobifida fusca. The riboswitch forms a classic H-type pseudoknot that includes a triple helix that is continuous with a central core of conserved nucleotides. These form a left-handed helical ramp of inter-nucleotide interactions, generating the guanidinium cation binding site. The ligand is hydrogen bonded to the Hoogsteen edges of two guanine bases. The binding pocket has a side opening that can accommodate a small side chain, shown by structures with bound methylguanidine, aminoguanidine, ethylguanidine, and agmatine. Comparison of the new structure with those of the guanidine I and II riboswitches reveals that evolution generated three different structural solutions for guanidine binding and subsequent gene regulation, although with some common elements., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The guanidine-II (mini-ykkC) riboswitch is the smallest of the guanidine-responsive riboswitches, comprising two stem loops of similar sequence. We have solved high-resolution crystal structures of both stem loops for the riboswitch from Gloeobacter violaceus. The stem loops have a strong propensity to dimerize by intimate loop-loop interaction. The dimerization creates specific binding pockets for two guanidine molecules, explaining their cooperative binding. Within the binding pockets the ligands are hydrogen bonded to a guanine at O6 and N7, and to successive backbone phosphates. Additionally they are each stacked upon a guanine nucleobase. One side of the pocket has an opening to the solvent, slightly lowering the specificity of ligand binding, and structures with bound methylguanidine, aminoguanidine, and agmatine show how this is possible., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Recent structural and mechanistic studies have shed considerable light on the catalytic mechanisms of nucleolytic ribozymes. The discovery of several new ribozymes in this class has now allowed comparisons to be made, and the beginnings of mechanistic groupings to emerge., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

The bright fluorescent cytosine analogue tC O stands out among fluorescent bases due to its virtually unquenched fluorescence emission in duplex DNA. However, like most reported base analogues, it has not been thoroughly characterized in RNA. We here report on the first synthesis and RNA-incorporation of tC O , and characterize its base-mimicking and fluorescence properties in RNA. As in DNA, we find a high quantum yield inside RNA duplexes (<Φ F > = 0.22) that is virtually unaffected by the neighbouring bases (Φ F  = 0.20-0.25), resulting in an average brightness of 1900 M -1 cm -1 . The average fluorescence lifetime in RNA duplexes is 4.3 ns and generally two lifetimes are required to fit the exponential decays. Fluorescence properties in ssRNA are defined by a small increase in average quantum yield (<Φ F  > = 0.24) compared to dsRNA, with a broader distribution (Φ F  = 0.17-0.34) and slightly shorter average lifetimes. Using circular dichroism, we find that the tC O -modified RNA duplexes form regular A-form helices and in UV-melting experiments the stability of the duplexes is only slightly higher than that of the corresponding natural RNA (<ΔT m > = + 2.3 °C). These properties make tC O a highly interesting fluorescent RNA base analogue for detailed FRET-based structural measurements, as a bright internal label in microscopy, and for fluorescence anisotropy measurements of RNA dynamics.

The TS ribozyme (originally called "twister sister") is a catalytic RNA. We present a crystal structure of the ribozyme in a pre-reactive conformation. Two co-axial helical stacks are organized by a three-way junction and two tertiary contacts. Five divalent metal ions are directly coordinated to RNA ligands, making important contributions to the RNA architecture. The scissile phosphate lies in a quasihelical loop region that is organized by a network of hydrogen bonding. A divalent metal ion is directly bound to the nucleobase 5' to the scissile phosphate, with an inner-sphere water molecule positioned to interact with the O2' nucleophile. The rate of ribozyme cleavage correlated in a log-linear manner with divalent metal ion pK a , consistent with proton transfer in the transition state, and we propose that the bound metal ion is a likely general base for the cleavage reaction. Our data indicate that the TS ribozyme functions predominantly as a metalloenzyme.

Holliday junction-resolving enzymes are nucleases that are highly specific for the structure of the junction, to which they bind in dimeric form. Two symmetrically disposed cleavages are made. These are not simultaneous, but the second cleavage is accelerated relative to the first, so ensuring that bilateral cleavage occurs during the lifetime of the DNA-protein complex. In eukaryotic cells there are two known junction-resolving activities. GEN1 is similar to enzymes from lower organisms. A crystallographic structure of a fungal GEN1 bound to the product of resolution has been determined. These complexes are dimerized within the crystal lattice such that the strands of the products may be simply reconnected to form a junction. These structures suggest a trajectory for the resolution process., (© 2016 Federation of European Biochemical Societies.)

Riboswitches are widespread in the bacterial kingdom, where they regulate gene expression in response to different small molecules and ions. However, some remain orphans with no known endogenous ligand. Three recently published papers (Battaglia et al., 2017; Nelson et al., 2017; and Reiss et al., 2017) de-orphanize a widespread ykkC element, showing that it responds to guanidine and elaborating on how that response is mediated., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Kink turns (k-turns) are widespread structural elements that introduce an axial bend into duplex RNA with an included angle of 50°. These mediate key tertiary interactions and bind specific proteins including members of the L7Ae family. The standard k-turn comprises a three-nucleotide bulge followed by G·A and A·G pairs. The RNA kinks by an association of the two minor grooves, stabilized by the formation of a number of key cross-strand hydrogen bonds mostly involving the adenine bases of the G·A and A·G pairs. The k-turns may be divided into two conformational classes, depending on the receptor for one of these hydrogen bonds. k-turns become folded by one of three different processes. Some, but not all, k-turns become folded in the presence of metal ions. Whether or not a given k-turn is folded under these conditions is determined by its sequence. We present a set of rules for the prediction of folding properties and the structure adopted on local sequence., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We present the crystal structure of the junction-resolving enzyme GEN1 bound to DNA at 2.5 Å resolution. The structure of the GEN1 protein reveals it to have an elaborated FEN-XPG family fold that is modified for its role in four-way junction resolution. The functional unit in the crystal is a monomer of active GEN1 bound to the product of resolution cleavage, with an extensive DNA binding interface for both helical arms. Within the crystal lattice, a GEN1 dimer interface juxtaposes two products, whereby they can be reconnected into a four-way junction, the structure of which agrees with that determined in solution. The reconnection requires some opening of the DNA structure at the center, in agreement with permanganate probing and 2-aminopurine fluorescence. The structure shows that a relaxation of the DNA structure accompanies cleavage, suggesting how second-strand cleavage is accelerated to ensure productive resolution of the junction., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Processing of Holliday junctions is essential in recombination. We have identified the gene for the junction-resolving enzyme GEN1 from the thermophilic fungus Chaetomium thermophilum and expressed the N-terminal 487-amino-acid section. The protein is a nuclease that is highly selective for four-way DNA junctions, cleaving 1nt 3' to the point of strand exchange on two strands symmetrically disposed about a diagonal axis. CtGEN1 binds to DNA junctions as a discrete homodimer with nanomolar affinity. Analysis of the kinetics of cruciform cleavage shows that cleavage of the second strand occurs an order of magnitude faster than the first cleavage so as to generate a productive resolution event. All these properties are closely similar to those described for bacterial, phage and mitochondrial junction-resolving enzymes. CtGEN1 is also similar in properties to the human enzyme but lacks the problems with aggregation that currently prevent detailed analysis of the latter protein. CtGEN1 is thus an excellent enzyme with which to engage in biophysical and structural analysis of eukaryotic GEN1., (Copyright © 2014. Published by Elsevier Ltd.)