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Large RNAs often utilize GNRA tetraloops as structural elements to stabilize the overall tertiary fold. These tetraloop-receptor (TR) interactions have a conserved geometry in which the tetraloop docks into the receptor at an angle of ~15° from the helix containing the receptor. Here, we show that the conserved GUAAY pentaloop found in domain III of group IIB1 introns participates in a novel class of RNA tertiary interaction with a geometry and mode of binding that are significantly different from that found in GNRA TR interactions. This pentaloop is highly conserved within the IIB1 class and interacts with the minor groove of the catalytic domain V. The base planes of the loop and receptor nucleotides are not coplanar and greatly deviate from standard A-minor motifs. The helical axis of the GUAAY stem loop diverges ~70° from the angle of insertion found in a typical GNRA TR interaction. Therefore, the loop architecture and insertion orientation are distinctive, with in vitro splicing data indicating that a GNRA tetraloop is incompatible at this position. The GUAAY pentaloop-receptor motif is also found in the structure of the eukaryotic thiamine pyrophosphate riboswitch in the context of a hexanucleotide loop sequence. We therefore propose, based on phylogenetic, structural, and biochemical data, that the GUAAY pentaloop-receptor interaction represents a novel structural motif that is present in multiple structured RNAs.

[Display omitted] • RNA structure-based approach reveals the intron landscape within human pathogenic fungi. • Group I introns are ubiquitously present in fungal pathogens, including all top-ranked human pathogens. • Representative introns from two major Candida pathogens catalyze highly efficient self-splicing reactions. Systemic fungal infections are a growing public health threat, and yet viable antifungal drug targets are limited as fungi share a similar proteome with humans. However, features of RNA metabolism and the noncoding transcriptomes in fungi are distinctive. For example, fungi harbor highly structured RNA elements that humans lack, such as self-splicing introns within key housekeeping genes in the mitochondria. However, the location and function of these mitochondrial riboregulatory elements has largely eluded characterization. Here we used an RNA-structure-based bioinformatics pipeline to identify the group I introns interrupting key mitochondrial genes in medically relevant fungi, revealing their fixation within a handful of genetic hotspots and their ubiquitous presence across divergent phylogenies of fungi, including all highest priority pathogens such as Candida albicans, Candida auris, Aspergillus fumigatus and Cryptococcus neoformans. We then biochemically characterized two representative introns from C. albicans and C. auris , demonstrating their exceptionally efficient splicing catalysis relative to previously-characterized group I introns. Indeed, the C. albicans mitochondrial intron displays extremely rapid catalytic turnover, even at ambient temperatures and physiological magnesium ion concentrations. Our results unmask a significant new set of players in the RNA metabolism of pathogenic fungi, suggesting a promising new type of antifungal drug target. [ABSTRACT FROM AUTHOR]

We have obtained a 1.55-Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni under conditions that permit detailed observations of Na(+) ion binding in the ribozyme's active site. At least two such Na(+) ions are observed. The first Na(+) ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical with that previously observed for divalent cations. A second Na(+) ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with that of previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na(+), but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na(+) directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active-site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest-resolution ribozyme structure in the Protein Data Bank.

L1 ligase (L1L) molecular switch is an in vitro optimized synthetic allosteric ribozyme that catalyzes the regioselective formation of a 5'-to-3' phosphodiester bond, a reaction for which there is no known naturally occurring RNA catalyst. L1L serves as a proof of principle that RNA can catalyze a critical reaction for prebiotic RNA self-replication according to the RNA world hypothesis. L1L crystal structure captures two distinct conformations that differ by a reorientation of one of the stems by around 80Å and are presumed to correspond to the active and inactive state, respectively. It is of great interest to understand the nature of these two states in solution and the pathway for their interconversion. In this study, we use explicit solvent molecular simulation together with a novel enhanced sampling method that utilizes concepts from network theory to map out the conformational transition between active and inactive states of L1L. We find that the overall switching mechanism can be described as a three-state/two-step process. The first step involves a large-amplitude swing that reorients stem C. The second step involves the allosteric activation of the catalytic site through distant contacts with stem C. Using a conformational space network representation of the L1L switch transition, it is shown that the connection between the three states follows different topographical patterns: the stem C swing step passes through a narrow region of the conformational space network, whereas the allosteric activation step covers a much wider region and a more diverse set of pathways through the network.

We have captured the structure of a pre-catalytic conformational intermediate of the hammerhead ribozyme using a phosphodiester tether formed between I and Stem II. This phosphodiester tether appears to mimic interactions in the wild-type hammerhead RNA that enable switching between nuclease and ligase activities, both of which are required in the replicative cycles of the satellite RNA viruses from which the hammerhead ribozyme is derived. The structure of this conformational intermediate reveals how the attacking nucleophile is positioned prior to cleavage, and demonstrates how restricting the ability of Stem I to rotate about its helical axis, via interactions with Stem II, can inhibit cleavage. Analogous covalent crosslinking experiments have demonstrated that imposing such restrictions on interhelical movement can change the hammerhead ribozyme from a nuclease to a ligase. Taken together, these results permit us to suggest that switching between ligase and nuclease activity is determined by the helical orientation of Stem I relative to Stem II.

Orbital steering is invoked to explain how the three-dimensional structure of a small self-cleaving RNA, the hammerhead ribozyme, both prevents and enhances RNA autocatalysis. Within the conserved catalytic core of the ribozyme, the position of the 2' oxygen atom of the G8 ribose is observed to be aligned almost perfectly with the phosphorus atom and the 5' oxygen atom of the adjacent A9 phosphate group for self-cleavage via an in-line attack mechanism. Despite this apparent near-perfect atomic positioning, no cleavage takes place. The explanation proposed is that a network of hydrogen bonds in the ribozyme core orients or steers the orbitals containing the electron lone pairs of the attacking nucleophile (the 2' oxygen atom) away from the A9 phosphorus atom, eliminating overlap with the vacant phosphorus d-orbitals despite the near-perfect in-line positioning of the oxygen atom, thus preventing catalysis. Because of the near-perfect atomic positioning of the 2' oxygen atom relative to the phosphate group, orbital steering effects in this case are fortuitously uncoupled from conformational, distance and orientation effects, allowing an assessment of the catalytic power due purely to orbital steering. In contrast, a conformational change at the cleavage site is required to bring the 2' oxygen atom and the scissile phosphate group into atomic positions amenable to an in-line attack mechanism. In addition, the conformationally changed structure must then steer the lone-pair orbitals of the correctly positioned 2' oxygen atom toward the scissile phosphorus atom in order for cleavage to take place. We estimate that fulfillment of each of these two required changes may contribute separately an approximately 1000-fold rate enhancement, potentially accounting for a significant fraction of the catalytic power of this ribozyme. Orbital steering therefore appears to be a general phenomenon that may help to explain catalysis in both ribozymes and protein enzymes in a unified manner.

The thermostable Geobacillus stearothermophilus GsI–IIC intron is among the few bacterial group II introns found to proliferate to high copy number in its host genome. Here, we developed a bacterial genetic assay for retrohoming and biochemical assays for protein-dependent and self-splicing of GsI–IIC. We found that GsI–IIC, like other group IIC introns, retrohomes into sites having a 5′-exon DNA hairpin, typically from a bacterial transcription terminator, followed by short intron-binding sequences (IBSs) recognized by base pairing of exon-binding sequences (EBSs) in the intron RNA. Intron RNA insertion occurs preferentially but not exclusively into the parental lagging strand at DNA replication forks, using a nascent lagging strand DNA as a primer for reverse transcription. In vivo mobility assays, selections, and mutagenesis indicated that a variety of GC-rich DNA hairpins of 7–19 bp with continuous base pairs or internal elbow regions support efficient intron mobility and identified a critically recognized nucleotide (T-5) between the hairpin and IBS1, a feature not reported previously for group IIC introns. Neither the hairpin nor T-5 is required for intron excision or lariat formation during RNA splicing, but the 5′-exon sequence can affect the efficiency of exon ligation. Structural modeling suggests that the 5′-exon DNA hairpin and T-5 bind to the thumb and DNA-binding domains of GsI–IIC reverse transcriptase. This mode of DNA target site recognition enables the intron to proliferate to high copy number by recognizing numerous transcription terminators and then finding the best match for the EBS/IBS interactions within a short distance downstream. [ABSTRACT FROM AUTHOR]

Abstract: The ai5γ group II intron requires a protein cofactor to facilitate native folding in the cell. Yeast protein Mss116 greatly accelerates intron folding under near-physiological conditions both in vivo and in vitro. Although the effect of Mss116 on the kinetics of ai5γ ribozyme folding and catalysis has been extensively studied, the precise structural role and interaction sites of Mss116 have been elusive. Using Nucleotide Analog Interference Mapping to study the folding of splicing precursor constructs, we have identified specific intron functional groups that participate in Mss116-facilitated folding and we have determined their role in the folding mechanism. The data indicate that Mss116 stabilizes an early, obligate folding intermediate within intron domain 1, thereby laying the foundation for productive folding to the native state. In addition, the data reveal an important role for the IBS2 exon sequence and for the terminus of domain 6, during the folding of self-splicing group IIB intron constructs. [Copyright &y& Elsevier]

Abstract: Like other RNA molecules, RNase P RNA (RPR) is composed of domains, and these have different functions. Here, we provide data demonstrating that the catalytic (C) domain of Escherichia coli (Eco) RPR when separated from the specificity (S) domain mediates cleavage using various model RNA hairpin loop substrates. Compared to full-length Eco RPR, the rate constant, k obs, of cleavage for the truncated RPR (CP RPR) was reduced 30- to 13,000-fold depending on substrate. Specifically, the structural architecture of the −1/+73 played a significant role where a C−1/G+73 pair had the most dramatic effect on k obs. Substitution of A248 (E. coli numbering), positioned near the cleavage site in the RNase P–substrate complex, with G in the CP RPR resulted in 30-fold improvement in rate. In contrast, strengthening the interaction between the RPR and the 3′ end of the substrate only had a modest effect. Interestingly, although deleting the S-domain gave a reduction in the rate, it resulted in a less erroneous RPR with respect to cleavage site selection. These data support and extend our understanding of the coupling between the distal interaction between the S-domain and events at the active site. Our findings will also be discussed with respect to the structure of RPR derived from different organisms. [Copyright &y& Elsevier]

Abstract: The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension (CTE) induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionally important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein–protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends on ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the CTE results primarily from a steric block. Finally, we identified two conserved regions—one the previously noted post II region in the helicase core and the other in the CTE—that may help displace or sequester the opposite RNA strand during RNA unwinding. [Copyright &y& Elsevier]

Abstract: We report a set of atomistic folding/unfolding simulations for the hairpin ribozyme using a Monte Carlo algorithm. The hairpin ribozyme folds in solution and catalyzes self-cleavage or ligation via a specific two-domain structure. The minimal active ribozyme has been studied extensively, showing stabilization of the active structure by cations and dynamic motion of the active structure. Here, we introduce a simple model of tertiary-structure formation that leads to a phase diagram for the RNA as a function of temperature and tertiary-structure strength. We then employ this model to capture many folding/unfolding events and to examine the transition-state ensemble (TSE) of the RNA during folding to its active “docked” conformation. The TSE is compact but with few tertiary interactions formed, in agreement with single-molecule dynamics experiments. To compare with experimental kinetic parameters, we introduce a novel method to benchmark Monte Carlo kinetic parameters to docking/undocking rates collected over many single molecular trajectories. We find that topology alone, as encoded in a biased potential that discriminates between secondary and tertiary interactions, is sufficient to predict the thermodynamic behavior and kinetic folding pathway of the hairpin ribozyme. This method should be useful in predicting folding transition states for many natural or man-made RNA tertiary structures. [Copyright &y& Elsevier]

Abstract: The yeast DEAD-box protein Mss116p functions as a general RNA chaperone in splicing mitochondrial group I and group II introns. For most of its functions, Mss116p is thought to use ATP-dependent RNA unwinding to facilitate RNA structural transitions, but it has been suggested to assist in the folding of one group II intron (aI5γ) primarily by stabilizing a folding intermediate. Here we compare three aI5γ constructs: one with long exons, one with short exons, and a ribozyme construct lacking exons. The long exons result in slower splicing, suggesting that they misfold and/or stabilize nonnative intronic structures. Nevertheless, Mss116p acceleration of all three constructs depends on ATP and is inhibited by mutations that compromise RNA unwinding, suggesting similar mechanisms. Results of splicing assays and a new two-stage assay that separates ribozyme folding and catalysis indicate that maximal folding of all three constructs by Mss116p requires ATP-dependent RNA unwinding. ATP-independent activation is appreciable for only a subpopulation of the minimal ribozyme construct and not for constructs containing exons. As expected for a general RNA chaperone, Mss116p can also disrupt the native ribozyme, which can refold after Mss116p removal. Finally, using yeast strains with mitochondrial DNA containing only the single intron aI5γ, we show that Mss116p mutants promote splicing in vivo to degrees that correlate with their residual ATP-dependent RNA-unwinding activities. Together, our results indicate that, although DEAD-box proteins play multiple roles in RNA folding, the physiological function of Mss116p in aI5γ splicing includes a requirement for ATP-dependent local unfolding, allowing the conversion of nonfunctional RNA structure into functional RNA structure. [Copyright &y& Elsevier]

Abstract: Bacterial RNase P consists of one protein and one RNA [RNase P RNA (RPR)]. RPR can process tRNA precursors correctly in the absence of the protein. Here we have used model hairpin loop substrates corresponding to the acceptor, T-stem, and T-loop of a precursor tRNA to study the importance of the T-loop structure in RPR-alone reaction. T-stem/loop (TSL) interacts with a region in RPR [TSL binding site (TBS)], forming TSL/TBS interaction. Altering the T-loop structure affects both cleavage site selection and rate of cleavage at the correct site +1 and at the alternative site −1. The magnitude of variation depended on the structures of the T-loop and the TBS region, with as much as a 150-fold reduction in the rate of cleavage at +1. Interestingly, for one T-loop structure mutant, no difference in the rate at −1 was detected compared to cleavage of the substrate with an unchanged T-loop, indicating that, in this case, the altered T-loop structure primarily influences events required for efficient cleavage at the correct site +1. We also provide data supporting a functional link between a productive TSL/TBS interaction and events at the cleavage site. Collectively, our findings emphasize the interplay between separate regions upon formation of a productive RPR substrate that leads to efficient and accurate cleavage. These new data provide support for an induced-fit mechanism in bacterial RPR-mediated cleavage at the correct site +1. [Copyright &y& Elsevier]

Abstract: The hepatitis delta virus ribozyme is a small, self-cleaving RNA with a compact tertiary structure and buried active site that is important in the life cycle of the virus. The ribozyme''s function in nature is to cleave an internal phosphodiester bond and linearize concatemers during rolling circle replication. Crystal structures of the ribozyme have been solved in both pre-cleaved and post-cleaved (product) forms and reveal an intricate network of interactions that conspire to catalyze bond cleavage. In addition, extensive biochemical studies have been performed to work out a mechanism for bond cleavage in which C75 and a magnesium ion catalyze the reaction by general acid–base chemistry. One issue that has remained unclear in this ribozyme and in other ribozymes is the nature of long-distance communication between peripheral regions of the RNA and the buried active site. We performed molecular dynamics simulations on the hepatitis delta virus ribozyme in the product form and assessed communication between a distal structural portion of the ribozyme—the protonated C41 base triple—and the active site containing the critical C75. We varied the ionization state of C41 in both the wild type and a C41 double mutant variant and determined the impact on the active site. In all four cases, effects at the active site observed in the simulations agree with experimental studies on ribozyme activity. Overall, these studies indicate that small functional RNAs have the potential to communicate interactions over long distances and that wild-type RNAs may have evolved ways to prevent such interactions from interfering with catalysis. [Copyright &y& Elsevier]

Abstract: Multiple studies hypothesize that DEAD-box proteins facilitate folding of the ai5γ group II intron. However, these conclusions are generally inferred from splicing kinetics, and not from direct monitoring of DEAD-box protein-facilitated folding of the intron. Using native gel electrophoresis and dimethyl sulfate structural probing, we monitored Mss-116-facilitated folding of ai5γ intron ribozymes and a catalytically active self-splicing RNA containing full-length intron and short exons. We found that the protein directly stimulates folding of these RNAs by accelerating formation of the compact near-native state. This process occurs in an ATP-independent manner, although ATP is required for the protein turnover. As Mss 116 binds RNA nonspecifically, most binding events do not result in the formation of the compact state, and ATP is required for the protein to dissociate from such nonproductive complexes and rebind the unfolded RNA. Results obtained from experiments at different concentrations of magnesium ions suggest that Mss 116 stimulates folding of ai5γ ribozymes by promoting the formation of unstable folding intermediates, which is then followed by a cascade of folding events resulting in the formation of the compact near-native state. Dimethyl sulfate probing results suggest that the compact state formed in the presence of the protein is identical to the near-native state formed more slowly in its absence. Our results also indicate that Mss 116 does not stabilize the native state of the ribozyme, but that such stabilization results from binding of attached exons. [Copyright &y& Elsevier]

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

Abstract: The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are broadly acting RNA chaperones that function in mitochondria to stimulate group I and group II intron splicing and to activate mRNA translation. Previous studies showed that the S. cerevisiae cytosolic/nuclear DEAD-box protein Ded1p could stimulate group II intron splicing in vitro. Here, we show that Ded1p complements mitochondrial translation and group I and group II intron splicing defects in mss116Δ strains, stimulates the in vitro splicing of group I and group II introns, and functions indistinguishably from CYT-19 to resolve different nonnative secondary and/or tertiary structures in the Tetrahymena thermophila large subunit rRNA-ΔP5abc group I intron. The Escherichia coli DEAD-box protein SrmB also stimulates group I and group II intron splicing in vitro, while the E. coli DEAD-box protein DbpA and the vaccinia virus DExH-box protein NPH-II gave little, if any, group I or group II intron splicing stimulation in vitro or in vivo. The four DEAD-box proteins that stimulate group I and group II intron splicing unwind RNA duplexes by local strand separation and have little or no specificity, as judged by RNA-binding assays and stimulation of their ATPase activity by diverse RNAs. In contrast, DbpA binds group I and group II intron RNAs nonspecifically, but its ATPase activity is activated specifically by a helical segment of E. coli 23S rRNA, and NPH-II unwinds RNAs by directional translocation. The ability of DEAD-box proteins to stimulate group I and group II intron splicing correlates primarily with their RNA-unwinding activity, which, for the protein preparations used here, was greatest for Mss116p, followed by Ded1p, CYT-19, and SrmB. Furthermore, this correlation holds for all group I and group II intron RNAs tested, implying a fundamentally similar mechanism for both types of introns. Our results support the hypothesis that DEAD-box proteins have an inherent ability to function as RNA chaperones by virtue of their distinctive RNA-unwinding mechanism, which enables refolding of localized RNA regions or structures without globally disrupting RNA structure. [Copyright &y& Elsevier]

Abstract: An in vitro selection search for DNAs capable of catalyzing photochemistry yielded two distinctive deoxyribozymes (DNAzymes) with photolyase activity: UV1C, which repaired thymine dimers within DNA using a UV light of >300 nm wavelength and no extraneous cofactor, and Sero1C, which required the tryptophan metabolite serotonin as cofactor in addition to the UV light. Catalysis by Sero1C conformed to Michaelis–Menten kinetics, and analysis of the action spectrum of Sero1C confirmed that serotonin did indeed serve as a catalytic cofactor rather than as a structural cofactor. Sero1C and UV1C showed strikingly distinct wavelength optima for their respective photoreactivation catalyses. Although the rate enhancements characteristic of the two DNAzymes were similar, the cofactor-requiring Sero1C repaired a substantially broader range of substrates compared to UV1C, including thymine, uracil, and a range of chimeric deoxypyrimidine and ribopyrimidine dimers. Similarities and differences in the properties of these two photolyase DNAzymes suggest, first, that the harnessing of less damaging UV light for the repair of photolesions may have been a primordial catalytic activity of nucleic acids, and, second, the broader substrate range of Sero1C may highlight an evolutionary advantage to coopting amino-acid-like cofactors by functionality-poor nucleic acid enzymes. [Copyright &y& Elsevier]

Abstract: Stable RNAs must fold into specific three-dimensional structures to be biologically active, yet many RNAs form metastable structures that compete with the native state. Our previous time-resolved footprinting experiments showed that Azoarcus group I ribozyme forms its tertiary structure rapidly (τ<30 ms) without becoming significantly trapped in kinetic intermediates. Here, we use stopped-flow fluorescence spectroscopy to probe the global folding kinetics of a ribozyme containing 2-aminopurine in the loop of P9. The modified ribozyme was catalytically active and exhibited two equilibrium folding transitions centered at 0.3 and 1.6 mM Mg2+, consistent with previous results. Stopped-flow fluorescence revealed four kinetic folding transitions with observed rate constants of 100, 34, 1, and 0.1 s−1 at 37 °C. From comparison with time-resolved Fe(II)-ethylenediaminetetraacetic acid footprinting of the modified ribozyme under the same conditions, these folding transitions were assigned to formation of the IC intermediate, tertiary folding and docking of the nicked P9 tetraloop, reorganization of the P3 pseudoknot, and refolding of nonnative conformers, respectively. The footprinting results show that 50–60% of the modified ribozyme folds in less than 30 ms, while the rest of the RNA population undergoes slow structural rearrangements that control the global folding rate. The results show how small perturbations to the structure of the RNA, such as a nick in P9, populate kinetic folding intermediates that are not observed in the natural ribozyme. [Copyright &y& Elsevier]

Abstract: Tetraloops are a common building block for RNA tertiary structure, and most tetraloops fall into one of three well-characterized classes: GNRA, UNCG, and CUYG. Here, we present the sequence and structure of a fourth highly conserved class of tetraloop that occurs only within the ζ–ζ′ interaction of group IIC introns. This GANC tetraloop was identified, along with an unusual cognate receptor, in the crystal structure of the group IIC intron and through phylogenetic analysis of intron RNA sequence alignments. Unlike conventional tetraloop–receptor interactions, which are stabilized by extensive hydrogen-bonding interactions, the GANC–receptor interaction is limited to a single base stack between the conserved adenosine of the tetraloop and a single purine of the receptor, which consists of a one- to three-nucleotide bulge and does not contain an A-platform. Unlike GNRA tetraloops, the GANC tetraloop forms a sharp angle relative to the adjacent helix, bending by approximately 45° toward the major groove side of the helix. These structural attributes allow GANC tetraloops to fit precisely within the group IIC intron core, thereby demonstrating that structural motifs can adapt to function in a specific niche. [Copyright &y& Elsevier]

Abstract: To understand whether 5′ and 3′ structural elements of the region corresponding to the mature tRNA affect the expression of the tRNA, we examined several bacterial genomes for tRNA genes where the expression might be potentially affected by structural elements located outside of the mature tRNA. In Pseudomonas aeruginosa, our analysis suggested that the tRNATrp is transcribed together with a putative stem–loop structure followed by a uridine tract immediately downstream of the tRNA region. This structural element, resembling a Rho-independent transcription terminator, might therefore influence the expression and processing of tRNATrp. Moreover, the secondary structure suggested that the discriminator base in the tRNATrp precursor can pair with either the C at position −1, the 3′ terminal residue in the 5′ leader, or the C immediately 5′ of the uridine tract of the putative Rho-independent transcription terminator. Here, we present in vivo data demonstrating the importance of residue −1 and the positioning of the putative transcription terminator for the expression of correctly 5′ processed P. aeruginosa tRNATrp in Escherichia coli. Interestingly, we also detected a difference in the appearance of correctly 5′ processed P. aeruginosa tRNATrp in E. coli compared to the situation in P. aeruginosa. [Copyright &y& Elsevier]

Abstract: The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are general RNA chaperones that function in splicing mitochondrial group I and group II introns and in translational activation. Both proteins consist of a conserved ATP-dependent RNA helicase core region linked to N and C-terminal domains, the latter with a basic tail similar to many other DEAD-box proteins. In CYT-19, this basic tail was shown to contribute to non-specific RNA binding that helps tether the core helicase region to structured RNA substrates. Here, multiple sequence alignments and secondary structure predictions indicate that CYT-19 and Mss116p belong to distinct subgroups of DEAD-box proteins, whose C-terminal domains have a defining extended α-helical region preceding the basic tail. We find that mutations or C-terminal truncations in the predicted α-helical region of Mss116p strongly inhibit RNA-dependent ATPase activity, leading to loss of function in both translational activation and RNA splicing. These findings suggest that the α-helical region may stabilize and/or regulate the activity of the RNA helicase core. By contrast, a truncation that removes only the basic tail leaves high RNA-dependent ATPase activity and causes only a modest reduction in translation and RNA splicing efficiency in vivo and in vitro. Biochemical analysis shows that deletion of the basic tail leads to weaker non-specific binding of group I and group II intron RNAs, and surprisingly, also impairs RNA-unwinding at saturating protein concentrations and nucleotide-dependent tight binding of single-stranded RNAs by the RNA helicase core. Together, our results indicate that the two sub-regions of Mss116p''s C-terminal domain act in different ways to support and modulate activities of the core helicase region, whose RNA-unwinding activity is critical for both the translation and RNA splicing functions. [Copyright &y& Elsevier]

Abstract: The D135 group II intron ribozyme follows a unique folding pathway that is direct and appears to be devoid of kinetic traps. During the earliest stages of folding, D135 collapses slowly to a compact intermediate, and all subsequent assembly events are rapid. Collapse of intron domain 1 (D1) has been shown to limit the rate constant for D135 folding, although the specific substructure of the D1 kinetic intermediate has not yet been identified. Employing time-resolved nucleotide analog interference mapping, we have identified a cluster of atoms within the D1 main stem that control the rate constant for D135 collapse. Functional groups within the κ–ζ element are particularly important for this earliest stage of folding, which is intriguing given that this same motif also serves later as the docking site for catalytic domain 5. More important, the κ–ζ element is shown to be a divalent ion binding pocket, indicating that this region is a Mg2+-dependent switch that initiates the cascade of D135 folding events. By measuring the Mg2+ dependence of the compaction rate constant, we conclude that the actual rate-limiting step in D1 compaction involves the formation of an unstable folding intermediate that is captured by the binding of Mg2+. This carefully orchestrated folding pathway, in which formation of an active-site docking region is early and rate limiting, ensures proper folding of the intron core and faithful splicing. It may represent an important paradigm for the folding of large, multidomain RNA molecules. [Copyright &y& Elsevier]

Abstract: RNase P with its catalytic RNA subunit is involved in the processing of a number of RNA precursors with different structures. However, precursor tRNAs are the most abundant substrates for RNase P. Available data suggest that a tRNA is folded into its characteristic structure already at the precursor state and that RNase P recognizes this structure. The tRNA D-/T-loop domain (TSL-region) is suggested to interact with the specificity domain of RNase P RNA while residues in the catalytic domain interact with the cleavage site. Here, we have studied the consequences of a productive interaction between the TSL-region and its binding site (TBS) in the specificity domain using tRNA precursors and various hairpin-loop model substrates. The different substrates were analyzed with respect to cleavage site recognition, ground-state binding, cleavage as a function of the concentration of Mg2+ and the rate of cleavage under conditions where chemistry is suggested to be rate limiting using wild-type Escherichia coli RNase P RNA, M1 RNA, and M1 RNA variants with structural changes in the TBS-region. On the basis of our data, we conclude that a productive TSL/TBS interaction results in a conformational change in the M1 RNA substrate complex that has an effect on catalysis. Moreover, it is likely that this conformational change comprises positioning of chemical groups (and Mg2+) at and in the vicinity of the cleavage site. Hence, our findings are consistent with an induced-fit mechanism in RNase P RNA-mediated cleavage. [Copyright &y& Elsevier]

Abstract: Metal ions play key roles in the folding and function for many structured RNAs, including group I introns. We determined the X-ray crystal structure of the Azoarcus bacterial group I intron in complex with its 5′ and 3′ exons. In addition to 222 nucleotides of RNA, the model includes 18 Mg2+ and K+ ions. Five of the metals bind within 12 Å of the scissile phosphate and coordinate the majority of the oxygen atoms biochemically implicated in conserved metal–RNA interactions. The metals are buried deep within the structure and form a multiple metal ion core that is critical to group I intron structure and function. Eight metal ions bind in other conserved regions of the intron structure, and the remaining five interact with peripheral structural elements. Each of the 18 metals mediates tertiary interactions, facilitates local bends in the sugar-phosphate backbone or binds in the major groove of helices. The group I intron has a rich history of biochemical efforts aimed to identify RNA–metal ion interactions. The structural data are correlated to the biochemical results to further understand the role of metal ions in group I intron structure and function. [Copyright &y& Elsevier]

Abstract: pK a calculations based on the Poisson–Boltzmann equation have been widely used to study proteins and, more recently, DNA. However, much less attention has been paid to the calculation of pK a shifts in RNA. There is accumulating evidence that protonated nucleotides can stabilize RNA structure and participate in enzyme catalysis within ribozymes. Here, we calculate the pK a shifts of nucleotides in RNA structures using numerical solutions to the Poisson–Boltzmann equation. We find that significant shifts are predicted for several nucleotides in two catalytic RNAs, the hairpin ribozyme and the hepatitis delta virus ribozyme, and that the shifts are likely to be related to their functions. We explore how different structural environments shift the pK as of nucleotides from their solution values. RNA structures appear to use two basic strategies to shift pK as: (a) the formation of compact structural motifs with structurally-conserved, electrostatic interactions; and (b) the arrangement of the phosphodiester backbone to focus negative electrostatic potential in specific regions. [Copyright &y& Elsevier]

Abstract: The folding of group II intron ribozymes has been studied extensively under optimal conditions for self-splicing in vitro (42 °C and high magnesium ion concentrations). In these cases, the ribozymes fold directly to the native state by an apparent two-state mechanism involving the formation of an obligate intermediate within intron domain 1. We have now characterized the folding pathway under near-physiological conditions. We observe that compaction of the RNA proceeds slowly to completion, even at low magnesium concentration (3 mM). Kinetic analysis shows that this compact species is a “near-native” intermediate state that is readily chased into the native state by the addition of high salt. Structural probing reveals that the near-native state represents a compact domain 1 scaffold that is not yet docked with the catalytic domains (D3 and D5). Interestingly, native ribozyme reverts to the near-native state upon reduction in magnesium concentration. Therefore, while the intron can sustain the intermediate state under physiological conditions, the native structure is not maintained and is likely to require stabilization by protein cofactors in vivo. [Copyright &y& Elsevier]

Abstract: Among the unexpected chemistries that can be catalyzed by nucleic acid enzymes is photochemistry. We have reported the in vitro selection of a small, cofactor-independent deoxyribozyme, UV1C, capable of repairing thymine dimers in a DNA substrate, most optimally with light at a wavelength of >300 nm. We hypothesized that a guanine quadruplex functioned both as a light antenna and an electron source for the repair of the substrate within the enzyme–substrate complex. Here, we report structural and mechanistic investigations of that hypothesis. Contact-crosslinking and guanosine to inosine mutational studies reveal that the thymine dimer and the guanine quadruplex are positioned close to each other in the deoxyribozyme–substrate complex, and permit us to refine the structure and topology of the folded deoxyribozyme. In exploring the substrate utilization capabilities of UV1C, we find it to be able to repair uracil dimers as well as thymine dimers, as long as they are present in an overall deoxyribonucleotide milieu. Some surprising similarities with bacterial CPD photolyase enzymes are noted. [Copyright &y& Elsevier]

Abstract: The RNA-catalyzed splicing of group I and group II introns is facilitated by proteins that stabilize the active RNA structure or act as RNA chaperones to disrupt stable inactive structures that are kinetic traps in RNA folding. In Neurospora crassa and Saccharomyces cerevisiae, the latter function is fulfilled by specific DEAD-box proteins, denoted CYT-19 and Mss116p, respectively. Previous studies showed that purified CYT-19 stimulates the in vitro splicing of structurally diverse group I and group II introns, and uses the energy of ATP binding or hydrolysis to resolve kinetic traps. Here, we purified Mss116p and show that it has RNA-dependent ATPase activity, unwinds RNA duplexes in a non-polar fashion, and promotes ATP-independent strand-annealing. Further, we show that Mss116p binds RNA non-specifically and promotes in vitro splicing of both group I and group II intron RNAs, as well as RNA cleavage by the aI5γ-derived D135 ribozyme. However, Mss116p also has ATP hydrolysis-independent effects on some of these reactions, which are not shared by CYT-19 and may reflect differences in its RNA-binding properties. We also show that a non-mitochondrial DEAD-box protein, yeast Ded1p, can function almost as efficiently as CYT-19 and Mss116p in splicing the yeast aI5γ group II intron and less efficiently in splicing the bI1 group II intron. Together, our results show that Mss116p, like CYT-19, can act broadly as an RNA chaperone to stimulate the splicing of diverse group I and group II introns, and that Ded1p also has an RNA chaperone activity that can be assayed by its effect on splicing mitochondrial introns. Nevertheless, these DEAD-box protein RNA chaperones are not completely interchangeable and appear to function in somewhat different ways, using biochemical activities that have likely been tuned by coevolution to function optimally on specific RNA substrates. [Copyright &y& Elsevier]

Most tRNAs carry a G at their 5′ termini, i.e. at position +1. This position corresponds to the position immediately downstream of the site of cleavage in tRNA precursors. Here we studied RNase P RNA-mediated cleavage of substrates carrying substitutions/modifications at position +1 in the absence of the RNase P protein, C5, to investigate the role of G at the RNase P cleavage site. We present data suggesting that the exocyclic amine (2NH2) of G+1 contributes to cleavage site recognition, ground state binding and catalysis by affecting the rate of cleavage. This is in contrast to O6, N7 and 2′OH that are suggested to affect ground state binding and rate of cleavage to significantly lesser extent. We also provide evidence that the effects caused by the absence of 2NH2 at position +1 influenced the charge distribution and conceivably Mg2+ binding at the RNase P cleavage site. These findings are consistent with models where the 2NH2 at the cleavage site (when present) interacts with RNase P RNA and/or influences the positioning of Mg2+ in the vicinity of the cleavage site. Moreover, our data suggest that the presence of the base at +1 is not essential for cleavage but its presence suppresses miscleavage and dramatically increases the rate of cleavage. Together our findings provide reasons why most tRNAs carry a guanosine at their 5′ end. [Copyright &y& Elsevier]

At the heart of the RNA folding problem is the number, structures, and relationships among the intermediates that populate the folding pathways of most large RNA molecules. Unique insight into the structural dynamics of these intermediates can be gleaned from the time-dependent changes in local probes of macromolecular conformation (e.g. reports on individual nucleotide solvent accessibility offered by hydroxyl radical ( OH) footprinting). Local measures distributed around a macromolecule individually illuminate the ensemble of separate changes that constitute a folding reaction. Folding pathway reconstruction from a multitude of these individual measures is daunting due to the combinatorial explosion of possible kinetic models as the number of independent local measures increases. Fortunately, clustering of time progress curves sufficiently reduces the dimensionality of the data so as to make reconstruction computationally tractable. The most likely folding topology and intermediates can then be identified by exhaustively enumerating all possible kinetic models on a super-computer grid. The folding pathways and measures of the relative flux through them were determined for Mg2+ and Na+-mediated folding of the Tetrahymena thermophila group I intron using this combined experimental and computational approach. The flux during Mg2+-mediated folding is divided among numerous parallel pathways. In contrast, the flux during the Na+-mediated reaction is predominantly restricted through three pathways, one of which is without detectable passage through intermediates. Under both conditions, the folding reaction is highly parallel with no single pathway accounting for more than 50% of the molecular flux. This suggests that RNA folding is non-sequential under a variety of different experimental conditions even at the earliest stages of folding. This study provides a template for the systematic analysis of the time-evolution of RNA structure from ensembles of local measures that will illuminate the chemical and physical characteristics of each step in the process. The applicability of this analysis approach to other macromolecules is discussed. [Copyright &y& Elsevier]

A library containing as many as 1016 nucleic acid candidates is typically used to isolate artificial ribozymes and deoxyribozymes (DNAzymes) in an in vitro selection experiment, with only a handful of sequences surviving many rounds of stringent selection steps. These winning species are generally the focus of interest whereas the less competitive contenders are usually not examined. Nevertheless, molecular species abandoned during the selection process might still represent a rich pool of catalytic motifs that are useful for the examination of DNA''s inherent catalytic ability, and for the design of molecular tools for practical applications. Here we report a study of six RNA-cleaving, fluorescence-signaling deoxyribozymes that appeared in the early generations of a previous in vitro selection experiment, using the combined approaches of reselection, rational structural analysis, and reaction condition optimization. All six deoxyribozymes were found to use a three-way junction as a common structural framework for catalysis. However, disparities observed in the conserved nucleotide allocations, methylation interference patterns and metal-ion selectivities, pointed to distinct catalytic cores. The rate constants of the optimized deoxyribozymes fell in the range of ∼0.2 to 1.6min−1, which are comparable to those of similar ribozymes. Our findings indicate that deoxyribozymes eliminated by harsh selection criteria are structurally simple molecules that can be tailored into efficient catalysts. [Copyright &y& Elsevier]

Large RNAs collapse into compact intermediates in the presence of counterions before folding to the native state. We previously found that collapse of a bacterial group I ribozyme correlates with the formation of helices within the ribozyme core, but occurs at Mg2+ concentrations too low to support stable tertiary structure and catalytic activity. Here, using small-angle X-ray scattering, we show that Mg2+-induced collapse is a cooperative folding transition that can be fit by a two-state model. The Mg2+ dependence of collapse is similar to the Mg2+ dependence of helix assembly measured by partial ribonuclease T1 digestion and of an unfolding transition measured by UV hypochromicity. The correspondence between multiple probes of RNA structure further supports a two-state model. A mutation that disrupts tertiary contacts between the L9 tetraloop and its helical receptor destabilized the compact state by 0.8kcal/mol, while mutations in the central triplex were less destabilizing. These results show that native tertiary interactions stabilize the compact folding intermediates under conditions in which the RNA backbone remains accessible to solvent. [Copyright &y& Elsevier]

RNase P mediated cleavage of the tRNAHis precursor does not rely on the formation of the “+73/294 interaction” to give the correct cleavage product, i.e. cleavage at −1, while other tRNA precursors that are cleaved at the canonical site +1 do. A previous model, here referred to as the “2′OH-model”, predicts that the 2′OH at the canonical cleavage site would affect cleavage at −1. Here we used model RNA hairpin substrates mimicking the structural architecture of the tRNAHis precursor cleavage site to investigate the role of 2′OH with respect to ground state binding and rate of cleavage in the presence and absence of the +73/294 interaction. Our data emphasize the importance of the 2′OH in the immediate vicinity of the scissile bond. Moreover, introduction of 2′H at the cleavage site did not affect cleavage at an alternative cleavage site to any significant extent. Our findings are therefore inconsistent with the 2′OH model. We favor a model where the 2′OH at the cleavage site influence Mg2+ binding in its vicinity, however we do not exclude the possibility that the 2′OH at the cleavage site interacts with RNase P RNA. Studying the importance of the 2′OH at different cleavage sites also indicated a higher dependence on the 2′OH at the cleavage site in the absence of the +73/294 interaction than in its presence. Finally, we provide data suggesting that N3 of U at position −1 in the substrate is most likely not involved in an interaction with RNase P RNA. [Copyright &y& Elsevier]

Fidelity in tRNA processing by the RNase P RNA from Escherichia coli depends, in part, on interactions with the nucleobase and 2′ hydroxyl group of N(−1), the nucleotide immediately upstream of the site of RNA strand cleavage. Here, we report a series of biochemical and structure–function studies designed to address how these interactions contribute to cleavage site selection. We find that simultaneous disruption of cleavage site nucleobase and 2′ hydroxyl interactions results in parallel reactions leading to correct cleavage and mis-cleavage one nucleotide upstream (5′) of the correct site. Changes in Mg2+ concentration and pH can influence the fraction of product that is incorrectly processed, with pH effects attributable to differences in the rate-limiting steps for the correct and mis-cleavage reaction pathways. Additionally, we provide evidence that interactions with the 2′ hydroxyl group adjacent to the reactive phosphate group also contribute to catalysis at the mis-cleavage site. Finally, disruption of the adjacent 2′-hydroxyl contact has a greater effect on catalysis when pairing between the ribozyme and N(−1) is also disrupted, and the effects of simultaneously disrupting these contacts on binding are also non-additive. One implication of these results is that mis-cleavage will result from any combination of active site modifications that decrease the rate of correct cleavage beyond a certain threshold. Indeed, we find that inhibition of correct cleavage and corresponding mis-cleavage also results from disruption of any combination of active site contacts including metal ion interactions and conserved pairing interactions with the 3′ RCCA sequence. Such redundancy in interactions needed for maintaining fidelity may reflect the necessity for multiple substrate recognition in vivo. These studies provide a framework for interpreting effects of substrate modifications on RNase P cleavage fidelity and provide evidence for interactions with the nucleobase and 2′ hydroxyl group adjacent to the reactive phosphate group in the transition state. [Copyright &y& Elsevier]

The time-course of monovalent cation-induced folding of the L-21 Sca1 Tetrahymena thermophila ribozyme and a selected mutant was quantitatively followed using synchrotron X-ray (·OH) footprinting. Initiating folding by increasing the concentration of either Na+ or K+ to 1.5M from an initial condition of ∼0.008M Na+ at 42°C resulted in the complete formation of tertiary contacts within the P5abc subdomain and between the peripheral helices within the dead time of our measurements (k>50s-1). These results contrast with folding rates of 2–0.2s-1 previously observed for formation of these contacts in 10mM Mg2+ from the same initial condition. Thus, the initial formation of native tertiary contacts is inhibited by divalent but not monovalent cations. The native contacts within the catalytic core form without a detectable burst phase at rates of 0.4–1.0s-1 in a manner reminiscent of the Mg2+-dependent folding behavior, although tenfold faster. The tertiary interactions stabilizing the catalytic core interaction with P4–P6 and P2.1, as well as one of the protections internal for the P4–P6 domain, display progress curves with appreciable burst amplitudes and a phase comparable in rate to that of the catalytic core. That the slow folding of the ribozyme''s core is a consequence of the alt-P3 secondary structure is shown by the 100% burst phase amplitudes that are observed for folding of the U273A mutant ribozyme within which the native secondary structure (P3) is strengthened. Thus, formation of a misfolded intermediate(s) resulting from the alt-P3 secondary structure is independent of ion valency while the rate at which the respective intermediates are resolved is sensitive to ion valency. The overall portrait painted by these results is that ion valency differentially affects steps in the folding process and that folding in monovalent ion alone for the U273A mutant Tetrahymena ribozyme is fast and direct. [Copyright &y& Elsevier]

We describe light-induced switches for the catalytic activity of the small, RNA-cleaving 8-17 deoxyribozyme (DNAzyme), based on photochemically induced cis–trans isomerization of azobenzene (Az) moieties covalently tethered at various locations within the DNAzyme. Prior studies have shown that trans-azobenzene is able to stack comfortably within a DNA double helix, stabilizing it, while cis-azobenzene has a helix-destabilizing effect. We designed two classes of Az-modified 8-17 DNAzyme constructs, in each of which two azobenzene molecules substituted for nucleotides, either in the substrate-binding arm (SBA); or, within the catalytic core. Measurement of single-turnover kinetics for RNA cleavage revealed that in the SBA constructs E11 and E13, five- to sixfold higher catalytic rates were obtained when the reaction mixture was irradiated with visible light (favouring trans-Az) as compared to ultraviolet light (which promotes cis-Az), consistent with trans-Az in these constructs stabilizing the enzyme–substrate complex. Surprisingly, the reverse result was obtained with the catalytic core construct E17, where ultraviolet irradiation resulted in a five- to sixfold faster catalytic activity relative to visible light irradiation. The development of such light-responsive nucleic acid enzymes may open new possibilities of using light as the activating or repressing agent in the control of gene expression within living cells and organisms. [Copyright &y& Elsevier]

The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA that resides in the HDV genome and regulates its replication. The native fold of the ribozyme is complex, having two pseudoknots. Earlier work implicated four non-native pairings in slowing pseudoknot formation: Alt 1, Alt 2, Alt 3, and Alt P1. The goal of the present work was design of a kinetically simplified and maximally reactive construct for in vitro mechanistic and structural studies. The initial approach chosen was site-directed mutagenesis in which known alternative pairings were destabilized while leaving the catalytic core intact. Based on prior studies, the G11C/U27Δ double mutant was prepared. However, biphasic kinetics and antisense oligonucleotide response trends opposite those of the well-studied G11C mutant were observed suggesting that new alternative pairings with multiple registers, termed Alt X and Alt Y, had been created. Enzymatic structure mapping of oligonucleotide models supported this notion. This led to a model wherein Alt 2 and the phylogenetically conserved Alt 3 act as “folding guides”, facilitating folding of the major population of the RNA molecules by hindering formation of the Alt X and Alt Y registers. Attempts to eliminate the strongest of the Alt X pairings by rational design of a quadruple mutant only resulted in more complex kinetic behavior. In an effort to simultaneously destabilize multiple alternative pairings, studies were carried out on G11C/U27Δ in the presence of urea or increased monovalent ion concentration. Inclusion of physiological ionic strength allowed the goal of monophasic, fast-folding (kobs≈60 min-1) kinetics to be realized. To account for this, a model is developed wherein Na+, which destabilizes secondary and tertiary structures in the presence of Mg2+, facilitates native folding by destabilizing the multiple alternative secondary structures with a higher-order dependence. [Copyright &y& Elsevier]

Mobile group II introns encode proteins with both reverse transcriptase activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active structure of the intron RNA. Previous studies with the Lactococcus lactis Ll.LtrB intron suggested a model in which the intron-encoded protein binds first to a high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of its own coding region, and then makes additional contacts with conserved catalytic core regions to stabilize the active RNA structure. Here, we developed an Escherichia coli genetic assay that links the splicing of the Ll.LtrB intron to the expression of green fluorescent protein and used it to study the in vivo splicing of wild-type and mutant introns and to delineate regions of the maturase required for splicing. Our results show that the maturase functions most efficiently when expressed in cis from the same transcript as the intron RNA. In agreement with previous in vitro assays, we find that the high-affinity binding site in DIVa is required for efficient splicing of the Ll.LtrB intron in vivo, but in the absence of DIVa, 6–10% residual splicing occurs by the direct binding of the maturase to the catalytic core. Critical regions of the maturase were identified by statistically analyzing ratios of missense to silent mutations in functional LtrA variants isolated from a library generated by mutagenic PCR (“unigenic evolution”). This analysis shows that both the reverse transcriptase domain and domain X, which likely corresponds to the reverse transcriptase thumb, are required for RNA splicing, while the C-terminal DNA-binding and DNA endonuclease domains are not required. Within the reverse transcriptase domain, the most critical regions for maturase activity include parts of the fingers and palm that function in template and primer binding in HIV-1 reverse transcriptase, but the integrity of the reverse transcriptase active site is not required. Biochemical analysis of LtrA mutants indicates that the N terminus of the reverse transcriptase domain is required for high-affinity binding of the intron RNA, possibly via direct interaction with DIVa, while parts of domain X interact with conserved regions of the catalytic core. Our results support the hypothesis that the intron-encoded protein adapted to function in splicing by using, at least in part, interactions used initially to recognize the intron RNA as a template for reverse transcription. [Copyright &y& Elsevier]

Mobile group II introns are site-specific retroelements that use a novel mobility mechanism in which the excised intron RNA inserts directly into a DNA target site and is then reverse transcribed by the associated intron-encoded protein. Because the DNA target site is recognized primarily by base-pairing of the intron RNA with only a small number of positions recognized by the protein, it has been possible to develop group II introns into a new type of gene targeting vector (“targetron”), which can be reprogrammed to insert into desired DNA targets simply by modifying the intron RNA. Here, we used databases of retargeted Lactococcus lactis Ll.LtrB group II introns and a compilation of nucleotide frequencies at active target sites to develop an algorithm that predicts optimal Ll.LtrB intron-insertion sites and designs primers for modifying the intron to insert into those sites. In a test of the algorithm, we designed one or two targetrons to disrupt each of 28 Escherichia coli genes encoding DExH/D-box and DNA helicase-related proteins and tested for the desired disruptants by PCR screening of 100 colonies. In 21 cases, we obtained disruptions at frequencies of 1–80% without selection, and in six other cases, where disruptants were not identified in the initial PCR screen, we readily obtained specific disruptions by using the same targetrons with a retrotransposition-activated selectable marker. Only one DExH/D-box protein gene, secA, which was known to be essential, did not give viable disruptants. The apparent dispensability of DExH/D-box proteins in E. coli contrasts with the situation in yeast, where the majority of such proteins are essential. The methods developed here should permit the rapid and efficient disruption of any bacterial gene, the computational analysis provides new insight into group II intron target site recognition, and the set of E. coli DExH/D-box protein and DNA helicase disruptants should be useful for analyzing the function of these proteins. [Copyright &y& Elsevier]

Despite a growing literature on the folding of RNA, our understanding of tertiary folding in large RNAs derives from studies on a small set of molecular examples, with primary focus on group I introns and RNase P RNA. To broaden the scope of RNA folding models and to better understand group II intron function, we have examined the tertiary folding of a ribozyme (D135) that is derived from the self-splicing ai5γ intron from yeast mitochondria. The D135 ribozyme folds homogeneously and cooperatively into a compact, well-defined tertiary structure that includes all regions critical for active-site organization and substrate recognition. When D135 was treated with increasing concentrations of Mg2+ and then subjected to hydroxyl radical footprinting, similar Mg2+ dependencies were seen for internalization of all regions of the molecule, suggesting a highly cooperative folding behavior. In this work, we show that global folding and compaction of the molecule have the same magnesium dependence as the local folding previously observed. Furthermore, urea denaturation studies indicate highly cooperative unfolding of the ribozyme that is governed by thermodynamic parameters similar to those for forward folding. In fact, D135 folds homogeneously and cooperatively from the unfolded state to its native, active structure, thereby demonstrating functional reversibility in RNA folding. Taken together, the data are consistent with two-state folding of the D135 ribozyme, which is surprising given the size and multi-domain structure of the RNA. The findings establish that the accumulation of stable intermediates prior to formation of the native state is not a universal feature of RNA folding and that there is an alternative paradigm in which the folding landscape is relatively smooth, lacking rugged features that obstruct folding to the native state. [Copyright &y& Elsevier]

Group II introns are self-splicing RNA molecules that also behave as mobile genetic elements. The secondary structure of group II intron RNAs is typically described as a series of six domains that project from a central wheel. Most structural and mechanistic analyses of the intron have focused on domains 1 and 5, which contain the residues essential for catalysis, and on domain 6, which contains the branch-point adenosine. Domains 2 and 3 (D2, D3) have been shown to make important contributions to intronic activity; however, information about their function is quite limited. To elucidate the role of D2 and D3 in group II ribozyme catalysis, we built a series of multi-piece ribozyme constructs based on the ai5γ group II intron. These constructs are designed to shed light on the roles of D2 and D3 in some of the major reactions catalyzed by the intron: 5′-exon cleavage, branching, and substrate hydrolysis. Reactions with these constructs demonstrate that D3 stimulates the chemical rate constant of group II intron reactions, and that it behaves as a form of catalytic effector. However, D3 is unable to associate independently with the ribozyme core. Docking of D3 is mediated by a short duplex that is found at the base of D2. In addition to recruiting D3 into the core, the D2 stem directs the folding of the adjacent j2/3 linker, which is among the most conserved elements in the group II intron active site. In turn, the D2 stem contributes to 5′-splice site docking and ribozyme conformational change. Nucleotide analog interference mapping suggests an interaction between the D2 stem and D3 that builds on the known θ–θ′ interaction and extends it into D3. These results establish that D3 and the base of D2 are key elements of the group II intron core and they suggest a hierarchy for active-site assembly. [Copyright &y& Elsevier]

A fundamental question in RNA folding is the mechanism of thermodynamic stability. We investigated the equilibrium folding of a series of sequence variants in which one to three motifs of a 255-nucleotide mesophilic ribozyme were substituted with the corresponding motifs from its thermophilic homologue. Substitution of three crucial motifs individually or in groups results in a continual increase in the stability and folding cooperativity in a stepwise fashion. We find an unexpected relationship between stability and folding cooperativity. Without changing the folding cooperativity, RNAs having a similar native structure can only achieve moderate change in stability and likewise, without changing stability, RNAs having a similar native structure can only achieve moderate change in folding cooperativity. This intricate relationship must be included in the predictions of tertiary RNA stability. [Copyright &y& Elsevier]

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human cytomegalovirus (HCMV) protease (PR), a viral protein that is responsible for the processing of the viral capsid assembly protein. We showed that the constructed ribozyme cleaved the PR mRNA sequence efficiently in vitro. Moreover, a reduction of about 80% in the expression level of the protease and a reduction of about 100-fold in HCMV growth were observed in cells that expressed the ribozyme stably. In contrast, a reduction of less than 10% in the expression of viral protease and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Further examination of the antiviral effects of the ribozyme-mediated cleavage of PR mRNA indicates that (1) the proteolytic cleavage of the capsid assembly protein is inhibited significantly, and (2) the packaging of the viral genomic DNA into the CMV capsids is blocked. These observations are consistent with the notion that the protease functions to process the capsid assembly protein and is essential for viral capsid assembly. Moreover, our results indicate that the RNase P ribozyme-mediated cleavage specifically reduces the expression of the protease, but not other viral genes examined. Thus, M1GS ribozyme is highly effective in inhibiting HCMV growth by targeting the PR mRNA and may represent a novel class of general gene-targeting agents for the studies and treatment of infections caused by human viruses, including HCMV. [Copyright &y& Elsevier]

The folding pathway of the Tetrahymena ribozyme correlates inversely with the sequence distance between native interactions, or contact order. The rapidly folding P4–P6 domain has a low contact order, while the slowly folding P3–P7 region has a high contact order. To examine the role of topology and contact order in RNA folding, we screened for circular permutants of the ribozyme that retain catalytic activity. Permutants beginning in the P4–P6 domain fold 5 to 20 times more slowly than the wild-type ribozyme. By contrast, 50% of a permuted RNA that disjoins a non-native interaction in P3 folds tenfold faster than the wild-type ribozyme. Hence, the probability of rapidly folding to the native state depends on the topology of tertiary domains. [Copyright &y& Elsevier]

We have studied an interaction, the “73/294-interaction”, between residues 294 in M1 RNA (the catalytic subunit of Escherichia coli RNase P) and +73 in the tRNA precursor substrate. The 73/294-interaction is part of the “RCCA-RNase P RNA interaction”, which anchors the 3′ R+73CCA-motif of the substrate to M1 RNA (interacting residues underlined). Considering that in a large fraction of tRNA precursors residue +73 is base-paired to nucleotide −1 immediately 5′ of the cleavage site, formation of the 73/294-interaction results in exposure of the cleavage site. We show that the nature/orientation of the 73/294-interaction is important for cleavage site recognition and cleavage efficiency. Our data further suggest that this interaction is part of a metal ion-binding site and that specific chemical groups are likely to act as ligands in binding of Mg2+ or other divalent cations important for function. We argue that this Mg2+ is involved in metal ion cooperativity in M1 RNA-mediated cleavage. Moreover, we suggest that the 73/294-interaction operates in concert with displacement of residue −1 in the substrate to ensure efficient and correct cleavage. The possibility that the residue at −1 binds to a specific binding surface/pocket in M1 RNA is discussed. Our data finally rationalize why the preferred residue at position 294 in M1 RNA is U. [Copyright &y& Elsevier]

Mobile group II introns encode reverse transcriptases that bind specifically to the intron RNAs to promote both intron mobility and RNA splicing (maturase activity). Previous studies with the Lactococcus lactis Ll.LtrB intron suggested a model in which the intron-encoded protein (LtrA) binds first to a primary high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of the LtrA coding sequence, and then makes additional contacts with conserved regions of the intron to fold the RNA into the catalytically active structure. Here, we analyzed the DIVa binding site by iterative in vitro selection and in vitro mutagenesis. Our results show that LtrA binds to a small region at the distal end of DIVa that contains the ribosome-binding site and initiation codon of the LtrA open reading frame. The critical elements are in a small stem-loop structure emanating from a purine-rich internal loop, with both sequence and structure playing a role in LtrA recognition. The ribosome-binding site falls squarely within the LtrA-binding region and is sequestered directly by the binding of LtrA or by stabilization of the small stem-loop or both. Finally, by using LacZ fusions in Escherichia coli, we show that the binding of LtrA to DIVa down-regulates translation. This mode of regulation limits accumulation of the potentially deleterious intron-encoded protein and may facilitate splicing by halting ribosome entry into the intron. The recognition of the DIVa loop-stem-loop structure accounts, in part, for the intron specificity of group II intron maturases and has parallels in template-recognition mechanisms used by other reverse transcriptases. [Copyright &y& Elsevier]

We have previously employed an in vitro (genetic) selection procedure to select RNase P ribozyme variants for their activity in cleaving a mRNA substrate from a pool of ribozymes containing randomized sequences. In this study, one of the variants was used to target the overlapping region of the mRNAs encoding the major transcription regulatory proteins, IE1 and IE2, of human cytomegalovirus (HCMV). The ribozyme variant exhibited an enhanced substrate binding and rate of chemical cleavage, and was at least 25 times more efficient in cleaving the target mRNA in vitro than the ribozyme derived from the wild-type sequence. Our results provide the first direct evidence that a point mutation at nucleotide 86 of RNase P catalytic RNA from Escherichia coli (A 86 → C 86 ) increases the rate of chemical cleavage while another mutation at nucleotide 205 (G 205 → C 205 ) enhances substrate binding of the ribozyme. Moreover, the variant was also more effective in inhibiting IE1 and IE2 expression and HCMV growth in cultured cells. A reduction of more than 97 % in IE1 and IE2 expression and a reduction of 3000-fold in viral growth were observed in cells expressing the variant. Thus, RNase P ribozyme variant is highly effective in inhibiting HCMV gene expression and growth. Our results provide the direct evidence that increasing the rate of chemical cleavage and substrate-binding affinity of the ribozymes should lead to an improvement of their anti-HCMV efficacy. Moreover, our data also suggest that highly effective anti-HCMV ribozyme variants can be developed using genetic engineering approaches including in vitro selection. [ABSTRACT FROM AUTHOR]