Contributions and competition of Mg2+ and K+ in folding and stabilization of the Twister ribozyme.

Native folded and compact intermediate states of RNA typically involve tertiary structures in the presence of divalent ions such as Mg2+ in a background of monovalent ions. In a recent study, we have shown how the presence of Mg2+ impacts the transition from partially unfolded to folded states through a "push-pull" mechanism where the ion both favors and disfavors the sampling of specific phosphate-phosphate interactions. To further understand the ion atmosphere of RNA in folded and partially folded states results from atomistic umbrella sampling and oscillating chemical potential grand canonical Monte Carlo/molecular dynamics (GCMC/MD) simulations are used to obtain atomic-level details of the distributions of Mg2+ and K+ ions around Twister RNA. Results show the presence of 100 mM Mg2+ to lead to increased charge neutralization over that predicted by counterion condensation theory. Upon going from partially unfolded to folded states, overall charge neutralization increases at all studied ion concentrations that, while associated with an increase in the number of direct ion-phosphate interactions, is fully accounted for by the monovalent K+ ions. Furthermore, K+ preferentially interacts with purine N7 atoms of helical regions in partially unfolded states, thereby potentially stabilizing the helical regions. Thus, both secondary helical structures and formation of tertiary structures leads to increased counterion condensation, thereby stabilizing those structural features of Twister. Notably, it is shown that K+ can act as a surrogate for Mg2+ by participating in specific interactions with nonsequential phosphate pairs that occur in the folded state, explaining the ability of Twister to self-cleave at submillimolar Mg2+ concentrations.

Solvent viscosity facilitates replication and ribozyme catalysis from an RNA duplex in a model prebiotic process.

The RNA World hypothesis posits that RNA was once responsible for genetic information storage and catalysis. However, a prebiotic mechanism has yet to be reported for the replication of duplex RNA that could have operated before the emergence of polymerase ribozymes. Previously, we showed that a viscous solvent enables information transfer from one strand of long RNA duplex templates, overcoming 'the strand inhibition problem'. Here, we demonstrate that the same approach allows simultaneous information transfer from both strands of long duplex templates. An additional challenge for the RNA World is that structured RNAs (like those with catalytic activity) function poorly as templates in model prebiotic RNA synthesis reactions, raising the question of how a single sequence could serve as both a catalyst and as a replication template. Here, we show that a viscous solvent also facilitates the transition of a newly synthesized hammerhead ribozyme sequence from its inactive, duplex state to its active, folded state. These results demonstrate how fluctuating environmental conditions can allow a ribozyme sequence to alternate between acting as a template for replication and functioning as a catalyst, and illustrate the potential for temporally changing environments to enable molecular processes necessary for the origin of life.

Molecular crowding favors reactivity of a human ribozyme under physiological ionic conditions.

In an effort to relate RNA folding to function under cellular-like conditions, we monitored the self-cleavage reaction of the human hepatitis delta virus-like CPEB3 ribozyme in the background of physiological ionic concentrations and various crowding and cosolute agents. We found that at physiological free Mg(2+) concentrations (∼0.1-0.5 mM), both crowders and cosolutes stimulate the rate of self-cleavage, up to ∼6-fold, but that in 10 mM Mg(2+) (conditions widely used for in vitro ribozyme studies) these same additives have virtually no effect on the self-cleavage rate. We further observe a dependence of the self-cleavage rate on crowder size, wherein the level of rate stimulation is diminished for crowders larger than the size of the unfolded RNA. Monitoring effects of crowding and cosolute agents on rates in biological amounts of urea revealed additive-promoted increases at both low and high Mg(2+) concentrations, with a maximal stimulation of more than 10-fold and a rescue of the rate to its urea-free values. Small-angle X-ray scattering experiments reveal a structural basis for this stimulation in that higher-molecular weight crowding agents favor a more compact form of the ribozyme in 0.5 mM Mg(2+) that is essentially equivalent to the form under standard ribozyme conditions of 10 mM Mg(2+) without a crowder. This finding suggests that at least a portion of the rate enhancement arises from favoring the native RNA tertiary structure. We conclude that cellular-like crowding supports ribozyme reactivity by favoring a compact form of the ribozyme, but only under physiological ionic and cosolute conditions.

Effects of chain length of polyethylene glycol molecular crowders on a mutant Tetrahymena group I ribozyme lacking large peripheral module.

While current group I ribozymes use several distinct strategies to function under conditions of low Mg2+ concentration (≤ 3 mM), a deletion mutant of the Tetrahymena ribozyme (ΔP5 ribozyme) is virtually inactive with 3 mM Mg2+ due to removal of the large peripheral module, P5abc, supporting the active conformation of the core module. We investigated the molecular crowding effects of synthetic polyethylene glycols (PEGs) on the activity of the ΔP5 ribozyme. Among PEG molecules with different chain lengths, PEG600 improved the activity of the ΔP5 ribozyme most effectively in the presence of 3 mM Mg2+.

Ribozymes and riboswitches: modulation of RNA function by small molecules.

Diverse small molecules interact with catalytic RNAs (ribozymes) as substrates and cofactors, and their intracellular concentrations are sensed by gene-regulatory mRNA domains (riboswitches) that modulate transcription, splicing, translation, or RNA stability. Although recognition mechanisms vary from RNA to RNA, structural analyses reveal recurring strategies that arise from the intrinsic properties of RNA such as base pairing and stacking with conjugated heterocycles, and cation-dependent recognition of anionic functional groups. These studies also suggest that, to a first approximation, the magnitude of ligand-induced reorganization of an RNA is inversely proportional to the complexity of the riboswitch or ribozyme. How these small molecule binding-induced changes in RNA lead to alteration in gene expression is less well understood. While different riboswitches have been proposed to be under either kinetic or thermodynamic control, the biochemical and structural mechanisms that give rise to regulatory consequences downstream of small molecule recognition by RNAs mostly remain to be elucidated.

Bright and Early: Inhibiting Human Cytomegalovirus by Targeting Major Immediate-Early Gene Expression or Protein Function.

The human cytomegalovirus (HCMV), one of eight human herpesviruses, establishes lifelong latent infections in most people worldwide. Primary or reactivated HCMV infections cause severe disease in immunosuppressed patients and congenital defects in children. There is no vaccine for HCMV, and the currently approved antivirals come with major limitations. Most approved HCMV antivirals target late molecular processes in the viral replication cycle including DNA replication and packaging. "Bright and early" events in HCMV infection have not been exploited for systemic prevention or treatment of disease. Initiation of HCMV replication depends on transcription from the viral major immediate-early (IE) gene. Alternative transcripts produced from this gene give rise to the IE1 and IE2 families of viral proteins, which localize to the host cell nucleus. The IE1 and IE2 proteins are believed to control all subsequent early and late events in HCMV replication, including reactivation from latency, in part by antagonizing intrinsic and innate immune responses. Here we provide an update on the regulation of major IE gene expression and the functions of IE1 and IE2 proteins. We will relate this insight to experimental approaches that target IE gene expression or protein function via molecular gene silencing and editing or small chemical inhibitors.

Visualizing the higher order folding of a catalytic RNA molecule.

The higher order folding process of the catalytic RNA derived from the self-splicing intron of Tetrahymena thermophila was monitored with the use of Fe(II)-EDTA-induced free radical chemistry. The overall tertiary structure of the RNA molecule forms cooperatively with the uptake of at least three magnesium ions. Local folding transitions display different metal ion dependencies, suggesting that the RNA tertiary structure assembles through a specific folding intermediate before the catalytic core is formed. Enzymatic activity, assayed with an RNA substrate that is complementary to the catalytic RNA active site, coincides with the cooperative structural transition. The higher order RNA foldings produced by Mg(II), Ca(II), and Sr(II) are similar; however, only the Mg(II)-stabilized RNA is catalytically active. Thus, these results directly demonstrate that divalent metal ions participate in general folding of the ribozyme tertiary structure, and further indicate a more specific involvement of Mg(II) in catalysis.

Footprinting the sites of interaction of antibiotics with catalytic group I intron RNA.

Aminoglycoside inhibitors of translation have been shown previously to inhibit in vitro self-splicing by group I introns. Chemical probing of the phage T4-derived sunY intron shows that neomycin, streptomycin, and related antibiotics protected the N-7 position of G96, a universally conserved guanine in the binding site for the guanosine cofactor in the splicing reaction. The antibiotics also disrupted structural contacts that have been proposed to bring the 5' cleavage site of the intron into proximity to the catalytic core. In contrast, the strictly competitive inhibitors deoxyguanosine and arginine protected only the N-7 position of G96. Parallels between these results and previously observed protection of 16S ribosomal RNA by aminoglycosides raise the possibility that group I intron splicing and transfer RNA selection by ribosomes involve similar RNA structural motifs.

A tetracycline-dependent ribozyme switch allows conditional induction of gene expression in Caenorhabditis elegans.

The nematode Caenorhabditis elegans represents an important research model. Convenient methods for conditional induction of gene expression in this organism are not available. Here we describe tetracycline-dependent ribozymes as versatile RNA-based genetic switches in C. elegans. Ribozyme insertion into the 3'-UTR converts any gene of interest into a tetracycline-inducible gene allowing temporal and, by using tissue-selective promoters, spatial control of expression in all developmental stages of the worm. Using the ribozyme switches we established inducible C. elegans polyglutamine Huntington's disease models exhibiting ligand-controlled polyQ-huntingtin expression, inclusion body formation, and toxicity. Our approach circumvents the complicated expression of regulatory proteins. Moreover, only little coding space is necessary and natural promoters can be utilized. With these advantages tetracycline-dependent ribozymes significantly expand the genetic toolbox for C. elegans.

Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme.

Available evidence suggests that Mg2+ ions are involved in reactions catalyzed by hammerhead ribozymes. However, the activity in the presence of exclusively monovalent ions led us to question whether divalent metal ions really function as catalysts when they are present. We investigated ribozyme activity in the presence of high levels of Mg2+ ions and the effects of Li+ ions in promoting ribozyme activity. We found that catalytic activity increased linearly with increasing concentrations of Mg2+ ions and did not reach a plateau value even at 1 M Mg2+ ions. Furthermore, this dependence on Mg2+ ions was observed in the presence of a high concentration of Li+ ions. These results indicate that the Mg2+ ion is a very effective cofactor but that the affinity of the ribozyme for a specific Mg2+ ion is very low. Moreover, cleavage by the ribozyme in the presence of both Li+ and Mg2+ ions was more effective than expected, suggesting the existence of a new reaction pathway-a cooperative pathway-in the presence of these multiple ions, and the possibility that a Mg2+ ion with weak affinity for the ribozyme is likely to function in structural support and/or act as a catalyst.

Presence of a coordinated metal ion in a trans-acting antigenomic delta ribozyme.

We have investigated the cleavage induced by metal ions in an antigenomic form of a trans-acting delta ribozyme. A specific Mg(2+)-induced cleavage at position G(52)at the bottom of the P2 stem was observed to occur solely within catalytically active ribozyme-substrate complexes (i.e. those that performed the essential conformational transition step). Only the divalent cations which support catalytic activity permitted the detection of specific induced cleavages in this region. Using various mutant ribozymes and substrates, we demonstrated a correlation between enzymatic activity and the Mg(2+)-induced cleavage pattern. We show that the efficiency of the coordination of the magnesium to its binding site is related to the nature of the base pair in the middle of the P1 stem (i.e. Rz(23)-S(8)). Together with additional evidence from nuclease probing experiments that indicates the occurrence of a structural rearrangement involving the bottom of the P2 stem upon formation of the P1 helix, these results show that an intimate relationship exists between the folding and the catalytic activity of the delta ribozyme.

Identifying antimalarial compounds targeting dihydrofolate reductase-thymidylate synthase (DHFR-TS) by chemogenomic profiling.

The mode of action of many antimalarial drugs is unknown. Chemogenomic profiling is a powerful method to address this issue. This experimental approach entails disruption of gene function and phenotypic screening for changes in sensitivity to bioactive compounds. Here, we describe the application of reverse genetics for chemogenomic profiling in Plasmodium. Plasmodium falciparum parasites harbouring a transgenic insertion of the glmS ribozyme downstream of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene were used for chemogenomic profiling of antimalarial compounds to identify those which target DHFR-TS. DHFR-TS expression can be attenuated by exposing parasites to glucosamine. Parasites with attenuated DHFR-TS expression were significantly more sensitive to antifolate drugs known to target DHFR-TS. In contrast, no change in sensitivity to other antimalarial drugs with different modes of action was observed. Chemogenomic profiling was performed using the Medicines for Malaria Venture (Switzerland) Malaria Box compound library, and two compounds were identified as novel DHFR-TS inhibitors. We also tested the glmS ribozyme in Plasmodium berghei, a rodent malaria parasite. The expression of reporter genes with downstream glmS ribozyme could be attenuated in transgenic parasites comparable with that obtained in P. falciparum. The chemogenomic profiling method was applied in a P. berghei line expressing a pyrimethamine-resistant Toxoplasma gondii DHFR-TS reporter gene under glmS ribozyme control. Parasites with attenuated expression of this gene were significantly sensitised to antifolates targeting DHFR-TS, but not other drugs with different modes of action. In conclusion, these data show that the glmS ribozyme reverse genetic tool can be applied for identifying primary targets of antimalarial compounds in human and rodent malaria parasites.

Aminoglycoside-RNA interactions.

The structural and physico-chemical parameters promoting the binding of aminoglycosides to RNAs are becoming clear. The strength of the interaction is dominated by electrostatics, with the positively charged aminoglycosides displacing metal ions. Although aminoglycosides inhibit most known ribozymes, aminoglycosides or polyamines are able to catalyze specific RNA cleavage in the absence of metal ions.

Kinetics at a multifunctional RNA active site.

Kinetics of a self-capping RNA, Iso6, have been investigated to constrain the catalytic mechanism. The role of phosphates has been examined by varying the number of phosphates on the nucleophilic attacking group or on the RNA. While the number of phosphates in the nucleophile affects capping kinetics, only KM but not kcat is altered. The KM values for GMP, GDP, GTP and ppppG are 200, 11, 13 and 31 microM, respectively. A reaction product, pyrophosphate, is also found to strongly inhibit RNA activities through a competitive exchange mechanism with an apparent Ki of 200 nM. Uniquely strong binding of pyrophosphate supports the idea that capping originated by utilization of the initial pyrophosphate leaving group site for capping nucleophiles. In contrast to the nucleophile phosphate, change of 5' RNA terminus from triphosphate to tetraphosphate enhances the overall rate and kcat by 40%, with little effect on KM. Thus, only the leaving group appears to affect the rate of the chemical transformation. We propose two possible mechanisms that explain this apparent rate-limiting chemical step, either dissociation of pyrophosphate to form a metaphosphate monoester intermediate or formation of a circular phosphoramidate intermediate, using an internal RNA nitrogenous group. A single essential Ca ion is required for all activities.

Monitoring of the cooperative unfolding of the sunY group I intron of bacteriophage T4. The active form of the sunY ribozyme is stabilized by multiple interactions with 3' terminal intron components.

We have studied the mechanism by which the 3' terminal domain of the sunY intron of bacteriophage T4 activates the group I ribozyme core of this intron, from which it is separated by some 800 nucleotides. As shown by monitoring either UV absorbance or self-splicing reaction kinetics as a function of temperature, intron transcripts undergo highly cooperative unfolding/inactivation upon heating: the two methods yield similar estimates of the thermodynamic parameters associated with this process. Such cooperativity makes it possible in turn to assess the energetic contribution of specific interactions to the overall structure, by comparing the sensitivity to heat inactivation of molecules carrying various nucleotide substitutions. By combining this approach with chemical modification, we have probed several proven or putative interactions between the core and 3' terminal domain of the intron and conclude that the role of the 3' terminal domain is to stabilize the active form of the ribozyme. Interestingly, the P9.0 interaction, which brings 3' terminal nucleotides next to the core site that binds the guanosine cofactor of the self-splicing reaction, is now shown to be composed in fact of two distinct pairings. An isolated base-pair (P9.0a), involving a residue located only six nucleotides upstream of the 3' splice site, participates in the stabilization of the ribozyme and appears to persist during the second stage of self-splicing (exon ligation). In contrast, formation of the previously demonstrated P9.0b pairing, which involves the two penultimate intron nucleotides, contributes no additional stability and results in no detectable rearrangement of the core structure. Implications for the concept of a static ribozyme are discussed in the light of a slightly revised three-dimensional model of the sunY intron.

Structure-mapping of the hairpin ribozyme. Magnesium-dependent folding and evidence for tertiary interactions within the ribozyme-substrate complex.

We have used chemical modification analysis to probe the solution structure of the hairpin ribozyme. The modifying reagents dimethylsulfate, 1-cyclohexyl-N'-[2-(N-methylmorpholino) ethyl-carbodiimide-p-toluenesulfonate, kethoxal, diethylpyrocarbonate and (2,12-dimethyl-3,7,11,17- tetraazabicyclo [11.3.1]heptadeca-1(17),2,11,13,15-pentaenato) nickel(II) perchlorate were used to probe functional groups that participate in Watson-Crick and non-canonical base-pairs. Our results confirm the existence of four short helices (3 to 6 bp) within the ribozyme-substrate complex, and demonstrate that one intramolecular helix (helix 4) is comprised of three base-pairs rather than the previously suggested five. In the absence of magnesium, the ribozyme is observed to fold into its secondary structure. Upon addition of magnesium, a striking difference in chemical modification is observed, particularly at sites within the ribozyme's large internal loop (loop B) that are essential for catalytic function (bases 21 to 26). Moreover, magnesium-dependent folding clearly destabilizes an A-U base-pair in a region where a proposed bend is required to juxtapose the catalytically essential loops A and B. Upon addition of substrate, no changes are observed in the structure of helix 3, loop B or helix 4. However, strong protection of bases in the substrate-binding domain is observed, including those located across internal loop A. The modification data are consistent with the formation of a previously proposed tertiary structure motif within loop B that includes non-canonical G-A, A-U and A-A base-pairs, and that is identical with those identified by NMR analysis of loop E of 5 S rRNA and the sarcin/ricin loop of 28 S rRNA. Our results indicate that the hairpin ribozyme adopts a stable magnesium-dependent tertiary structure to which the substrate binds without inducing major conformational changes, and that substrate recognition is likely to involve non-canonical base-pairs between the ribozyme and substrate sequences adjacent to the cleavage site.

Function of P11, a tertiary base pairing in self-splicing introns of subgroup IA.

There is phylogenetic evidence for the existence of a new pairing in subgroup IA1 self-splicing introns. This tertiary interaction, called P11, which is extraneous to the catalytic centre of these ribozymes was modelled after a "pseudoknot" and grafted by computer modelling on the common core structure of group I introns that was recently proposed by Michel & Westhof. In order to probe the function of the P11 pairing, we mutated the P11 helix in the intron of the large ribosomal precursor of Saccharomyces cerevisiae mitochondria (Sc.LSU). Our experimental data show that the P11 pairing plays a role in stabilizing the overall fold of the RNA molecule. While P11 is not essential for self-splicing activity in vitro, mutants with disrupted P11 require higher concentration of MgCl2 for self-splicing. By contrast, mutants with a reinforced P11 pairing (via introduction of several G.C base-pairs) self-splice more efficiently than the wild-type at 55 degrees C. Based on this work, the possible engineering of new stable versions of the ribozyme is discussed.

Kinetics and thermodynamics of the RNase P RNA cleavage reaction: analysis of tRNA 3'-end variants.

We have studied the interaction of 3'-end variants of a (pre-)tRNAGly with ribonuclease P (RNase P) RNAs from Escherichia coli and Thermus thermophilus. To dissect the thermodynamics of tRNA binding from the overall catalytic reaction, specific binding of mature tRNAGly variants to RNase P RNAs was studied by gel retardation. A newly developed assay, based on the reduction of Pb(2+)-hydrolysis at the CCA end due to complex formation of tRNA and RNase P RNA, was utilized to confirm the dissociation constants. The binding data were supplemented by single and multiple turnover kinetic analyses of the corresponding pre-tRNAGly variants. For E. coli RNase P RNA the following results were obtained. Extensions of CCA by pCp or three nucleotides (AUA) stabilized gel-resolved tRNAGly binding by 1 to 1.5 kcal/mol. Changing the first C in CCA to A, G or U resulted in a more than 100-fold reduction in binding affinity, which corresponds to a loss of 3.5 to 4.5 kcal/mol of binding energy. However, single turnover rate constants were only slightly affected, indicating that a disruption or loss of the tRNA 3'-end-mediated interaction with RNase P RNA does not preferentially destabilize the transition state. Our data suggest another kinetic step following initial substrate binding to E. coli RNase P RNA (possibly a conformational rearrangement). For T. thermophilus RNase P RNA, product release of wild-type tRNAGly CCAAUA was not rate-limiting in the multiple turnover reaction. However, the effects of CCA mutations were similar to those attained with E. coli RNase P RNA. This supports the notion that a high-affinity binding site for the tRNA 3'-end is a ubiquitous feature of eubacterial P RNAs. Finally, the results obtained here provide further evidence that the gel retardation assay is suitable for binding interference studies to identify the structural elements of RNase P RNAs and tRNAs that are crucial for the formation of a specific RNase P RNA-tRNA complex.

Antibiotic-induced oligomerisation of group I intron RNA.

Antibiotics act as inhibitors of various biological processes. Here we demonstrate that some tuberactinomycins, hitherto known as inhibitors of prokaryotic protein synthesis and of group I intron self-splicing, have a modulatory effect on group I intron RNAs. The linear intron, which is excised during the self-splicing process, is still an active molecular capable of performing an intramolecular transesterification resulting in a circular molecule. However, in the presence of sub-inhibitory concentrations of tuberactinomycins, the intron reacts intermolecularly leading to the formation of linear head-to-tail intron-oligomers. The antibiotic stimulates the intron to react in trans instead of in cis. The phage T4-derived td intron uses the same sites for oligomerisation as for circularisation. Gel- retardation experiments demonstrate that the intron RNA forms non-covalent complexes in the presence of the antibiotic. It might be envisaged that the role of these peptide antibiotics is to bridge RNA molecules mediating RNA-RNA interactions and thus enabling their reaction. The tuberactinomycins are further able to induce the interaction of heterologous introns. The ligation of the T4 phage-derived td intron with the Tetrahymena rRNA intron is very efficient, resulting in molecules composed of two introns derived from different species. The td intron attacks the Tetrahymena intron at various sites, which are located within double-stranded regions. These observations suggest that small molecules like these basic peptide antibiotics could have mediated RNA-RNA interactions in a pre-protein era.

Mapping in three dimensions of regions in a catalytic RNA protected from attack by an Fe(II)-EDTA reagent.

The accessibility of the ribose groups in the phosphodiester chain of M1 RNA, the catalytic subunit of ribonuclease P from Escherichia coli, has been probed with an Fe(II)-EDTA reagent when the RNA is alone in solution, when it is in a complex with a tRNA precursor substrate, and when it is in the holoenzyme complex with its cofactor, C5 protein. The regions found to be protected under these various conditions, as well as those previously identified in other chemical probing experiments, have been mapped on a three-dimensional working model of M1 RNA and are generally compatible with the previously proposed placement of the substrate on the enzyme and with previous data and inferences regarding the interactions of C5 protein with M1 RNA. On the basis of the accessibilities of the C(4') atoms, refinements have been introduced in the model to accommodate the Fe(II)-EDTA protection data. The protein cofactor makes contact with several helical regions of the catalytic RNA on the opposite side of the surface to which substrates bind.