Cross-ligation and exchange reactions catalyzed by hairpin ribozymes.

The negative strand of the satellite RNA of tobacco ringspot virus (sTobRV(-)) contains a hairpin catalytic domain that shows self-cleavage and self-ligation activities in the presence of magnesium ions. We describe here that the minimal catalytic domain can catalyze a cross-ligation reaction between two kinds of substrates in trans. The cross-ligated product increased when the reaction temperature was decreased during the reaction from 37 degrees C to 4 degrees C. A two-stranded hairpin ribozyme, divided into two fragments between G45 and U46 in a hairpin loop, showed higher ligation activity than the nondivided ribozyme. The two stranded ribozyme also catalyzed an exchange reaction of the 3'-portion of the cleavage site.     

Catalytically active geometry in the reversible circularization of 'mini-monomer' RNAs derived from the complementary strand of tobacco ringspot virus satellite RNA.

The less abundant polarity of the satellite RNA of tobacco ringspot virus, designated sTobRV(-)RNA, contains a ribozyme and its substrate. We demonstrate that the ribozyme can catalyze the ligation of substrate cleavage products and that oligoribonucleotides, termed 'mini-monomers' and containing little more than covalently attached ribozyme and substrate cleavage products, circularized spontaneously, efficiently and reversibly. The kinetics of ligation and cleavage of one such mini-monomer was consistent with a simple unimolecular reaction at some temperatures. Evidence suggests that the circular ligation product includes a 5 bp stem that is connected to a 4 bp stem by a bulge loop. Reduction of the bulge loop to one nt is expected to place the 4 and 5 bp helices in a nearly coaxial, rather than an angled or parallel, orientation. Such molecules did not circularize in a unimolecular reaction but did when incubated with second, trans-acting oligoribonucleotides that had either the original or a substituted 4 bp helix. These results suggest that a bulge loop that is too small prevents formation of geometry essential for unimolecular ligation. We suggest the term 'paperclip' to represent the arrangement of RNA strands in the region of sTobRV(-)RNA that participates in the cleavage and ligation reactions.     

RNA-directed construction of structurally complex and active ligase ribozymes through recombination

RNA-directed recombination can be used to catalyze a disproportionation reaction among small RNA substrates to create new combinations of sequences. But the accommodation of secondary and tertiary structural constraints in the substrates by recombinase ribozymes has not been explored. Here, we show that the Azoarcus group I intron can recombine oligoribonucleotides to construct class I ligase ribozymes, which are catalytically active upon synthesis. The substrate oligonucleotides, ranging in size from 58 to 104 nucleotides (nt), along with the 152-nt ligase ribozymes they reconstitute, can contain significant amounts of secondary structure. However, substrate recognition by the Azoarcus ribozyme depends on the existence of a single accessible CAU triplet for effective recombination. A biphasic temperature reaction profile was designed such that the sequential recombination/ligation events could take place in a thermocycler without human intervention. A temperature-dependent pH shift of the reaction buffer contributes to the success of the net reaction. When the substrate for the ligase ribozyme is introduced into the reaction mixture, as much as 11% can be observed being converted to product by the recombined ligase in the same reaction vessel. Recombination followed by ligation can also occur under isothermal conditions at 37 degrees C. Tertiary structure formation of the ligase upon construction can provide some protection from cleavage by the Azoarcus ribozyme when compared to the constituent substrates. These data suggest that RNA-directed recombination can, in fact, articulate complex ribozymes, and that there are logical rules that can guide the optimal placement of the CAU recognition sequence.     

Kinetic framework for ligation by an efficient RNA ligase ribozyme

The class I RNA ligase ribozyme, isolated previously from random sequences, performs an efficient RNA ligation reaction. It ligates two substrate RNAs, promoting the attack of the 3'-hydroxyl of one substrate upon the 5'-triphosphate of the other substrate with release of pyrophosphate. This ligation reaction has similarities to the reaction catalyzed by RNA polymerases. Using data from steady-state kinetic measurements and pulse-chase/pH-jump experiments, we have constructed minimal kinetic frameworks for two versions of the class I ligase, named 207t and 210t. For both ligases, as well as for the self-ligating parent ribozyme, the rate constant for the chemical step (k(c)) is log-linear with pH in the range 5.7-8.0. At physiological pH, the k(c) is 100 min(-1), a value similar to those reported for the fastest naturally occurring ribozymes. At higher pH, product release is limiting for both 207t and 210t. The 210t ribozyme, with its faster product release, attains multiple-turnover rates (k(cat) = 360 min(-1), pH 9.0) exceeding those of 207t and other reported ribozyme reactions. The kinetic framework for the 210t ribozyme describes the limits of this catalysis and suggests how key steps can be targeted for improvement using design or combinatorial approaches.     

The three-dimensional architecture of the class I ligase ribozyme

The class I ligase ribozyme catalyzes a Mg(++)-dependent RNA-ligation reaction that is chemically analogous to a single step of RNA polymerization. Indeed, this ribozyme constitutes the catalytic domain of an accurate and general RNA polymerase ribozyme. The ligation reaction is also very rapid in both single- and multiple-turnover contexts and thus is informative for the study of RNA catalysis as well as RNA self-replication. Here we report the initial characterization of the three-dimensional architecture of the ligase. When the ligase folds, several segments become protected from hydroxyl-radical cleavage, indicating that the RNA adopts a compact tertiary structure. Ribozyme folding was largely, though not completely, Mg(++) dependent, with a K(1/2[Mg]) < 1 mM, and was observed over a broad temperature range (20 degrees C -50 degrees C). The hydroxyl-radical mapping, together with comparative sequence analyses and analogy to a region within 23S ribosomal RNA, were used to generate a three-dimensional model of the ribozyme. The predictive value of the model was tested and supported by a photo-cross-linking experiment.     

Cross-ligation and exchange reaction of RNA catalyzed by hairpin ribozymes.

The catalytic domain in the minus strand of the satellite RNA of tobacco ringspot virus (sTobRV(-)) assumes a hairpin-like secondary structure. This ribozyme catalyzes a cross-ligation reaction between substrate RNAs of different lengths. We constructed ribozymes to probe the activities of ligation and RNA fragment exchange.     

Efficient, pH-dependent RNA ligation by the VS ribozyme in trans

The VS ribozyme acts as a very efficient ligase in trans when the 5' cleavage product is prevented from dissociation by an extended helix Ia in the substrate. Provided that the length of this helix is >or=10 bp, the substrate becomes approximately 80% ligated by the ribozyme acting in trans. Most of the nucleotides that have been shown to be important for cleavage are similarly important for ligation, including the critical A756 of the active site. The exception to this is C755. The variant ribozyme C755A has almost normal cleavage activity, whereas the rate of ligation is reduced 70-fold. It is therefore likely that this nucleotide plays a specific role in the organization of the termini of the ligation substrates. We have found that the rate of the trans ligation reaction depends on pH, corresponding to the protonation/deprotonation of a group with a pK(A) of 5.6. A model is suggested whereby the approach to equilibrium is catalyzed by the ribozyme catalyzing the ligation reaction in its deprotonated state (rate 1.05 min(-1)) and the cleavage reaction in its protonated state (rate 0.18 min(-1)). A756 is a candidate for the nucleobase undergoing protonation/deprotonation.     

In vitro adaptation of a ligase ribozyme for activity under a low-pH condition.

A ligase ribozyme accelerating a ligation reaction with oligonucleotide under a low-pH condition was selected by in vitro adaptation. A ribozyme active at pH 7 was randomly mutated, and the resultant RNA library was subjected to in vitro adaptation under a low-pH reaction condition. At pH 4, the adapted RNAs reacted with the oligonucleotide substrates about 200 times faster than the original ribozyme. When the ribozyme was cloned and sequenced, 10 of the 30 clones sequenced had identical sequences. The differences in sequence from the original ribozyme were found at four positions in the middle region and at the 3' end. A few sequential differences dominated the activity of the ribozyme under the extreme condition. The adapted ribozyme had one repeating sequence that was critical for the activity.     

In vitro evolution and characterization of a ligase ribozyme adapted to acidic conditions: effect of further rounds of evolution

A ligase ribozyme that accelerates the ligation reaction with an oligonucleotide under low pH conditions was identified by in vitro adaptation in a previous study. We examined the effects of further rounds of evolution to isolate a more active ribozyme. The ribozyme, which was obtained after four rounds of evolution, was randomly mutated, and the resultant RNA library was subjected to in vitro selection at low pH. One ribozyme isolated from the pool was found to react 8,000 times faster than the original b1 ribozyme at pH 4. The reaction rate of the isolated ribozyme was enhanced at various pH values, and its pH dependence was less than that of the original ribozyme or the ribozyme selected with four rounds of evolution. The reaction rate of the isolated ribozyme was reduced in the presence of 3' primer, the sequence of which is complementary to the 3' primer-binding site of the ligase ribozyme. This inhibition induced by the primer oligonucleotide binding to the ribozyme 3' region implies that the 3' region plays a role in the ligation reaction of the ribozyme.     

Non-ribozyme sequences enhance self-cleavage of ribozymes derived from Hepatitis delta virus.

Analysis of the self-cleavage of ribozymes derived from the genomic RNA of Hepatitis delta virus (HDV) has revealed that certain co-transcribed vector sequences significantly affect the activity of the ribozyme. Specifically, the t1/2 of self-cleavage for a 135 nucleotide HDV RNA varied, at 42 degrees C, from 5 min to 88 min, depending on the vector-derived sequences flanking the 5' end of the ribozyme. Further analysis suggested that this phenomenon was most likely due to the interaction of vector-derived sequences with a 16 nucleotide region found at the 3' end of the ribozyme. These findings have implications for studies of ribozymes transcribed from cDNA templates, and may provide information regarding the catalytic structure of the HDV ribozyme.     

Hairpin ribozyme-catalyzed ligation in water-alcohol solutions

The hairpin ribozyme (HPR) is a naturally existing RNA that catalyzes site-specific RNA cleavage and ligation. At 37 degrees C and in the presence of divalent metal ions (M(2+)), the HPR efficiently cleaves RNA substrates in trans. Here, we show that the HPR can catalyze efficient M(2+)-independent ligation in trans in aqueous solutions containing any of several alcohols, including methanol, ethanol, and isopropanol, and millimolar concentrations of monovalent cations. Ligation proceeds most efficiently in 60% isopropanol at 37 degrees C, whereas the reverse (cleavage) reaction is negligible under these conditions. We suggest that dehydration of the RNA is the key factor promoting HPR activity in water- alcohol solutions. Alcohol-induced ribozyme ligation may have practical applications.     

The effect of cytidine on the structure and function of an RNA ligase ribozyme

A cytidine-free ribozyme with RNA ligase activity was obtained by in vitro evolution, starting from a pool of random-sequence RNAs that contained only guanosine, adenosine, and uridine. This ribozyme contains 74 nt and catalyzes formation of a 3',5'-phosphodiester linkage with a catalytic rate of 0.016 min(-1). The RNA adopts a simple secondary structure based on a three-way junction motif, with ligation occurring at the end of a stem region located several nucleotides away from the junction. Cytidine was introduced to the cytidine-free ribozyme in a combinatorial fashion and additional rounds of in vitro evolution were carried out to allow the molecule to adapt to this added component. The resulting cytidine-containing ribozyme formed a 3',5' linkage with a catalytic rate of 0.32 min(-1). The improved rate of the cytidine-containing ribozyme was the result of 12 mutations, including seven added cytidines, that remodeled the internal bulge loops located adjacent to the three-way junction and stabilized the peripheral stem regions.     

Directing the outcome of deoxyribozyme selections to favor native 3'-5' RNA ligation

Previous experiments have identified numerous RNA ligase deoxyribozymes, each of which can synthesize either 2',5'-branched RNA, linear 2'-5'-linked RNA, or linear 3'-5'-linked RNA. These products may be formed by reaction of a 2'-hydroxyl or 3'-hydroxyl of one RNA substrate with the 5'-triphosphate of a second RNA substrate. Here the inherent propensities for nucleophilic reactivity of specific hydroxyl groups were assessed using RNA substrates related to the natural sequences of spliceosome substrates and group II introns. With the spliceosome substrates, nearly half of the selected deoxyribozymes mediate a ligation reaction involving the natural branch-point adenosine as the nucleophile. In contrast, mostly linear RNA is obtained with the group II intron substrates. Because the two sets of substrates differ at only three nucleotides, we conclude that the location of the newly created ligation junction in DNA-catalyzed branch formation depends sensitively on the RNA substrate sequences. During the experiment that led primarily to branched RNA, we abruptly altered the selection strategy to demand that the deoxyribozymes create linear 3'-5' linkages by introducing an additional selection step involving the 3'-5'-selective 8-17 deoxyribozyme. Although no 3'-5' linkages (相似文献   

Sequences required for self-catalysed cleavage of the satellite RNA of tobacco ringspot virus

The satellite RNA of tobacco ringspot virus (sTobRV) undergoes self-catalysed cleavage during replication. A plasmid for in vitro expression of sTobRV has been constructed and used to obtain a library of mutagenized sTobRV sequences. Screening of these mutants has allowed precise definition of the sequences required for (+) and (-) strand cleavage. The sequences and RNA structures associated with cleavage of each strand differ markedly. Cleavage of the (+) strand requires those sequences flanking the site for cleavage to form a 'hammerhead' domain, similar to those found in other satellite and viroid RNA. In contrast, cleavage of the (-) strand requires only a small region of 12 nucleotides (nt) at the site of cleavage, and a sequence of 55 nt positioned elsewhere in the molecule. Comparison with a closely related satellite suggests that a novel RNA structure may be involved in (-) strand cleavage.     

Structure-function analysis of Methanobacterium thermoautotrophicum RNA ligase - engineering a thermostable ATP independent enzyme

ABSTRACT: BACKGROUND: RNA ligases are essential reagents for many methods in molecular biology including NextGen RNA sequencing. To prevent ligation of RNA to itself, ATP independent mutant ligases, defective in self-adenylation, are often used in combination with activated pre-adenylated linkers. It is important that these ligases not have de-adenylation activity, which can result in activation of RNA and formation of background ligation products. An additional useful feature is for the ligase to be active at elevated temperatures. This has the advantage or reducing preferences caused by structures of single-stranded substrates and linkers. RESULTS: To create an RNA ligase with these desirable properties we performed mutational analysis of the archaeal thermophilic RNA ligase from Methanobacterium thermoautotrophicum. We identified amino acids essential for ATP binding and reactivity but dispensable for phosphodiester bond formation with 5' pre-adenylated donor substrate. The motif V lysine mutant (K246A) showed reduced activity in the first two steps of ligation reaction. The mutant has full ligation activity with pre-adenylated substrates but retained the undesirable activity of deadenylation, which is the reverse of step 2 adenylation. A second mutant, an alanine substitution for the catalytic lysine in motif I (K97A) abolished activity in the first two steps of the ligation reaction, but preserved wild type ligation activity in step 3. The activity of the K97A mutant is similar with either pre-adenylated RNA or single-stranded DNA (ssDNA) as donor substrates but we observed two-fold preference for RNA as an acceptor substrate compared to ssDNA with an identical sequence. In contrast, truncated T4 RNA ligase 2, the commercial enzyme used in these applications, is significantly more active using pre-adenylated RNA as a donor compared to pre-adenylated ssDNA. However, the T4 RNA ligases are ineffective in ligating ssDNA acceptors. CONCLUSIONS: Mutational analysis of the heat stable RNA ligase from Methanobacterium thermoautotrophicum resulted in the creation of an ATP independent ligase. The K97A mutant is defective in the first two steps of ligation but retains full activity in ligation of either RNA or ssDNA to a pre-adenylated linker. The ability of the ligase to function at 65 deg C should reduce the constraints of RNA secondary structure in RNA ligation experiments.     

Self-Replication Reactions Dependent on Tertiary Interaction Motifs in an RNA Ligase Ribozyme

RNA can function both as an informational molecule and as a catalyst in living organisms. This duality is the premise of the RNA world hypothesis. However, one flaw in the hypothesis that RNA was the most essential molecule in primitive life is that no RNA self-replicating system has been found in nature. To verify whether RNA has the potential for self-replication, we constructed a new RNA self-assembling ribozyme that could have conducted an evolvable RNA self-replication reaction. The artificially designed, in vitro selected ligase ribozyme was employed as a prototype for a self-assembling ribozyme. The ribozyme is composed of two RNA fragments (form R1·Z1) that recognize another R1·Z1 molecule as their substrate and perform the high turnover ligation reaction via two RNA tertiary interaction motifs. Furthermore, the substrate recognition of R1·Z1 is tolerant of mutations, generating diversity in the corresponding RNA self-replicating network. Thus, we propose that our system implies the significance of RNA tertiary motifs in the early RNA molecular evolution of the RNA world.     

The secondary structure and sequence optimization of an RNA ligase ribozyme.

In vitro selection can generate functional sequence variants of an RNA structural motif that are useful for comparative analysis. The technique is particularly valuable in cases where natural variation is unavailable or non-existent. We report the extension of this approach to a new extreme--the identification of a 112 nt ribozyme secondary structure imbedded within a 186 nt RNA. A pool of 10(14) variants of an RNA ligase ribozyme was generated using combinatorial chemical synthesis coupled with combinatorial enzymatic ligation such that 172 of the 186 relevant positions were partially mutagenized. Active variants of this pool were enriched using an in vitro selection scheme that retains the sequence variability at positions very close to the ligation junction. Ligases isolated after four rounds of selection catalyzed self-ligation up to 700 times faster than the starting sequence. Comparative analysis of the isolates indicated that when complexed with substrate RNAs the ligase forms a nested, double pseudo-knot secondary structure with seven stems and several important joining segments. Comparative analysis also suggested the identity of mutations that account for the increased activity of the selected ligase variants; designed constructs incorporating combinations of these changes were more active than any of the individual ligase isolates.