Ribozyme Active Site Research Articles

The solution structure of the VS ribozyme active site loop reveals a dynamic "hot-spot".

The VS ribozyme is the largest ribozyme in its class and is also the least structurally characterized thus far. The current working model of the VS ribozyme locates the active site in stem-loop VI. The solution structure of this active site loop was determined using high resolution NMR spectroscopy. The structure reveals that the ground-state conformation of the active site differs significantly from that determined previously from chemical structure probing and mutational analysis of the ribozyme in its active conformation, which contains several looped out bases. In contrast, the base-pairing scheme found for the isolated loop contains three mismatched base-pairs: an A+-C, a G-U wobble, and a sheared G-A base-pair and no looped out bases. Dynamics observed within the active site loop provide insight into the mechanism by which the RNA can rearrange its secondary structure into an "activated" conformation prior to cleavage. These findings lend support to the idea that RNA secondary structure is more fluid than once believed and that a better understanding of structure and dynamic features of ribozymes is required to unravel the intricacies of their catalytic abilities.

Solution structure of domain 5 of a group II intron ribozyme reveals a new RNA motif.

Domain 5 (D5) is the central core of group II intron ribozymes. Many base and backbone substituents of this highly conserved hairpin participate in catalysis and are crucial for binding to other intron domains. We report the solution structures of the 34-nucleotide D5 hairpin from the group II intron ai5 gamma in the absence and presence of divalent metal ions. The bulge region of D5 adopts a novel fold, where G26 adopts a syn conformation and flips down into the major groove of helix 1, close to the major groove face of the catalytic AGC triad. The backbone near G26 is kinked, exposing the base plane of the adjacent A-U pair to the solvent and causing bases of the bulge to stack intercalatively. Metal ion titrations reveal strong Mg(2+) binding to a minor groove shelf in the D5 bulge. Another distinct metal ion-binding site is observed along the minor groove side of the catalytic triad, in a manner consistent with metal ion binding in the ribozyme active site.

In Vitro Selection of Second Site Revertants Analysis of the Hairpin Ribozyme Active Site

We have used in vitro genetics to evaluate the function and interactions of the conserved base G8 in the hairpin ribozyme catalytic RNA. Second site revertant selection for a G8X mutant, where X is any of the other three natural nucleobases, yielded a family of second site suppressors of the G8U mutant, but not of G8C or G8A, indicating that only G and U can be tolerated at position 8 of the ribozyme. This result is consistent with recent observations that point to the functional importance of G8 N-1 in the chemistry of catalysis by this ribozyme reaction. Suppression of the G8U mutation was observed when changes were made directly across loop A from the mutated base at substrate position +2 or positions +2 and +3 in combination. The same changes made in the context of the natural G8 sequence resulted in a very large drop in activity. Thus, the G8U mutation results in a change in specificity of the ribozyme from 5'-N / GUC-3' to 5'-N / GCU-3'. The results presented imply that G8 interacts directly with U+2 during catalysis. We propose that this interaction favors the correct positioning of the catalytic determinants of G8. The implications for the folding of the ribozyme and the catalytic mechanism are discussed.

The Role of Magnesium Ions and 2′-Hydroxyl Groups in the VS Ribozyme–Substrate Interaction

The minimal substrate of the trans-cleaving Neurospora VS ribozyme has a stem-loop structure and interacts with the ribozyme by RNA tertiary interactions that remain only partially defined. The magnesium ion dependence of the catalytic parameters of a trans-cleaving VS-derived ribozyme were studied. The turnover number of the catalytic RNA was found to depend on the binding of at least three magnesium ions, with an apparent magnesium ion dissociation constant of 16 mM, but K M was observed to be metal ion independent in the millimolar range. To address the role of 2′-hydroxyl groups of the VS substrate RNA in interactions with the ribozyme, 23 altered substrates, each with a single 2′-deoxyribonucleoside substitution, were synthesised and their kinetic properties in the VS ribozyme reaction were analysed. The removal of five 2′-hydroxyl groups, at positions G620, A621, U628, C629 and G630 inhibited the reaction, whereas at two sites, G623 and A639, reaction was stimulated by the modification. Substitution of G620 with a 2′-deoxynucleoside was expected to inhibit the reaction, in line with the critical role of this 2′-hydroxyl group in the transesterification reaction. Altered substrates in which a 2′- O-methyl nucleoside replaced A621, U628, C629 and G630 were prepared and characterised. Although removal of the hydroxyl group of A621 inhibited the turnover number of the ribozyme significantly, this activity was recovered upon 2′- O-methyl adenosine substitution, suggesting that the 2′-oxygen atom of this nucleoside forms an important contact within the ribozyme active site. A cluster of residues within the loop region of the substrate, were more modestly affected by 2′-deoxynucleoside substitution. In two cases, magnesium binding was impaired, suggesting that stem-loop I is a possible magnesium ion binding site.

Catalysis of amide synthesis by RNA phosphodiester and hydroxyl groups.

The functional groups found among the RNA bases and in the phosphoribose backbone represent a limited repertoire from which to construct a ribozyme active site. This work investigates the possibility that simple RNA phosphodiester and hydroxyl functional groups could catalyze amide bond synthesis. Reaction of amine groups with activated esters would be catalyzed by a group that stabilizes the partial positive charge on the amine nucleophile in the transition state. 2'-Amine substitutions adjacent to 3'-phosphodiester or 3'-hydroxyl groups react efficiently with activated esters to form 2'-amide and peptide products. In contrast, analogs in which the 3'-phosphodiester is replaced by an uncharged phosphotriester or is constrained in a distal conformation react at least 100-fold more slowly. Similarly, a nucleoside in which the 3'-hydroxyl group is constrained trans to the 2'-amine is also unreactive. Catalysis of synthetic reactions by RNA phosphodiester and ribose hydroxyl groups is likely to be even greater in the context of a preorganized and solvent-excluding catalytic center. One such group is the 2'-hydroxyl of the ribosome-bound P-site adenosine substrate, which is close to the amine nucleophile in the peptidyl synthesis reaction. Given ubiquitous 2'-OH groups in RNA, there exists a decisive advantage for RNA over DNA in catalyzing reactions of biological significance.

Characterization of an RNA active site: interactions between a Diels-Alderase ribozyme and its substrates and products.

Ribozymes have recently been shown to catalyze the stereoselective formation of carbon-carbon bonds between small organic molecules. The interactions of these Diels-Alderase ribozymes with their substrates and products have now been elucidated by chemical substitution analysis by using 44 different, systematically varied analogues. RNA-diene interaction is governed by stacking interactions, while hydrogen bonding and metal ion coordination appear to be less important. The diene has to be an anthracene derivative, and substituents at defined positions are permitted, thereby shedding light on the geometry of the binding site. The dienophile must be a five-membered maleimidyl ring with an unsubstituted reactive double bond, and a hydrophobic side chain makes a major contribution to RNA binding. The ribozyme distinguishes between different enantiomers of chiral substrates and accelerates cycloadditions with both enantio- and diastereoselectivity. The stereochemistry of the reaction is controlled by RNA-diene interactions. The RNA interacts strongly and stereoselectively with the cycloaddition products, requiring several structural features to be present. Taken together, the results highlight the intricacy of ribozyme active sites which can control chemical reaction pathways based on minute differences in substrate stereochemistry and substitution pattern.

Identification of an active site ligand for a group I ribozyme catalytic metal ion.

The transition state of the group I intron self-splicing reaction is stabilized by three metal ions. The functional groups within the intron substrates (guanosine and an oligoribonucleotide mimic of the 5'-exon) that coordinate these metal ions have been systematically defined through a series of metal ion specificity switch experiments. In contrast, the catalytic metal ligands within the ribozyme active site are unknown. In an effort to identify them, stereospecific (R(P) or S(P)) single-site phosphorothioate substitutions were introduced at five phosphates predicted to be in the vicinity of the catalytic center (A207, C208, A304, U305, and A306) within the Tetrahymena intron. Of the 10 ribozymes that were studied, four phosphorothioate substitutions (A207 S(P), C208 S(P), A306 R(P), and A306 S(P)) exhibited a significant reduction in the cleavage rate. Only the effect of the C208 S(P) phosphorothioate substitution could be significantly rescued by the addition of a thiophilic metal ion, either Mn(2+) or Zn(2+), when tested with an all-oxy substrate. The effect was not rescued with Cd(2+). To determine if one of the catalytic metal ions is coordinated to the C208 pro-S(P) oxygen, the phosphorothioate-substituted ribozymes were also assayed using oligonucleotide substrates with a 3'-phosphorothiolate or an S(P) phosphorothioate substitution at the scissile phosphate. This resulted in a second metal specificity switch, in that Mn(2+) or Zn(2+) no longer rescued the C208 S(P) ribozyme, but Cd(2+) provided efficient rescue in the context of either sulfur-containing substrate. The 3'-oxygen and the pro-S(P) oxygen of the scissile phosphate are both known to coordinate the same metal ion, M(A), which stabilizes the negative charge on the leaving group 3'-oxygen in the transition state. Taken together, these data suggest that metal M(A) is coordinated to the C208 pro-S(P) phosphate oxygen, which constitutes the first functional link between a specific catalytic metal ion and a particular functional group within the group I ribozyme active site.

SITE SPECIFIC INCORPORATION OF 6-AZAURIDINE INTO THE GENOMIC HDV RIBOZYME ACTIVE SITE

The HDV ribozyme is proposed to catalyze its self cleavage reaction by a proton transfer mechanism wherein the N3 of its C75 acts as a general acid. The C75 to U mutation, which raises the N3 pKa from about 4 to almost 10, abolishes all enzymatic activity. To test if a U analogue with a neutral pKa can restore ribozyme function we incorporated 6-azauridine (n6U), a uridine analogue with histidine-like N3 pKa, into the genomic HDV ribozyme active site by 2′-O-ACE oligoribonucleotide protection chemistry. The resulting ribozymes were analyzed for their ability to undergo the HDV ribozyme cis-cleavage reaction. Incorporation of n6U at nucleotide position 75 did not restore ribozyme function compared to the U75 mutant. This suggests that the HDV ribozyme reaction mechanism involves more than positioning of a neutral nucleobase at the active site and implies that the exocyclic amino group of C75 participates in establishing the proper active site fold.

Catalytic cleavage of cis- and trans-acting antigenomic delta ribozymes in the presence of various divalent metal ions.

Catalytic activity of four structural variants of the antigenomic delta ribozyme, two cis- and two trans-acting, has been compared in the presence of selected divalent metal ions that effectively support catalysis. The ribozymes differ in regions that are not directly involved in formation of the ribozyme active site: the region immediately preceding the catalytic cleavage site, the P4 stem and a stretch of the viral RNA sequence extending the minimal ribozyme sequence at its 3'-terminus. The variants show high cleavage activity in the presence of Mg(2+), Ca(2+) and Mn(2+), lower with Co(2+) and Sr(2+) and some variants are also active with Cd(2+) and Zn(2+) ions. In the presence of a particular metal ion the ribozymes cleave, however with different initial rates, according to pseudo-first or higher order kinetics and to different final cleavage extents. On the other hand, relatively small differences are observed in the reactions induced by various metal ions. The cleavage of trans-acting ribozymes induced by Mg(2+) is partially inhibited in the presence of Na(+), spermidine and some other divalent metal ions. The inert Co(NH(3))(6)(3+) complex is unable to support catalysis, as reported earlier for the genomic ribozyme. The results are discussed in terms of the influence of structural elements peripheral to the ribozyme active site on its cleavage rate and efficiency as well as the role of metal ions in the cleavage mechanism. Some implications concerning further studies and possible applications of delta ribozymes are also considered.

PII: S0969-2126(01)00614-1

PII: S0969-2126(01)00614-1

Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis.

The hairpin ribozyme catalyses sequence-specific cleavage of RNA. The active site of this natural RNA results from the docking of two irregular helices: stems A and B. One strand of stem A harbours the scissile bond. The 2.4 A resolution structure of a hairpin ribozyme-inhibitor complex reveals that the ribozyme aligns the 2'-OH nucleophile and the 5'-oxo leaving group by twisting apart the nucleotides that flank the scissile phosphate. The base of the nucleotide preceding the cleavage site is stacked within stem A; the next nucleotide, a conserved guanine, is extruded from stem A and accommodated by a highly complementary pocket in the minor groove of stem B. Metal ions are absent from the active site. The bases of four conserved purines are positioned potentially to serve as acid-base catalysts. This is the first structure determination of a fully assembled ribozyme active site that catalyses a phosphodiester cleavage without recourse to metal ions.

Adenine protonation in domain B of the hairpin ribozyme.

Protein enzymes often use ionizable side chains, such as histidine, for general acid-base catalysis because the imidazole pK(a) is near neutral pH. RNA enzymes, on the other hand, are comprised of nucleotides which do not have apparent pK(a) values near neutral pH. Nevertheless, it has been recently shown that cytidine and adenine protonation can play an important role in both nucleic acid structure and catalysis. We have employed heteronuclear NMR methods to determine the pK(a) values and time scales of chemical exchanges associated with adenine protonation within the catalytically essential B domain of the hairpin ribozyme. The large, adenine-rich internal loop of the B domain allows us to determine adenine pK(a) values for a variety of non-Watson-Crick base pairs. We find that adenines within the internal loop have pK(a) values ranging from 4.8 to 5.8, significantly higher than the free mononucleotide pK(a) of 3. 5. Adenine protonation results in potential charge stabilization, hydrogen bond formation, and stacking interactions that are expected to stabilize the internal loop structure at low pH. Fast proton exchange times of 10-50 micros were determined for the well-resolved adenines. These results suggest that shifted pK(a) values may be a common feature of adenines in non-Watson-Crick base pairs, and identify two adenines which may participate in hairpin ribozyme active site chemistry.

An important base triple anchors the substrate helix recognition surface within the Tetrahymena ribozyme active site.

Key to understanding the structural biology of catalytic RNA is determining the underlying networks of interactions that stabilize RNA folding, substrate binding, and catalysis. Here we demonstrate the existence and functional importance of a Hoogsteen base triple (U300.A97-U277), which anchors the substrate helix recognition surface within the Tetrahymena group I ribozyme active site. Nucleotide analog interference suppression analysis of the interacting functional groups shows that the U300.A97-U277 triple forms part of a network of hydrogen bonds that connect the P3 helix, the J8/7 strand, and the P1 substrate helix. Product binding and substrate cleavage kinetics experiments performed on mutant ribozymes that lack this base triple (C A-U, U G-C) or replace it with the isomorphous C(+).G-C triple show that the A97 Hoogsteen triple contributes to the stabilization of both substrate helix docking and the conformation of the ribozyme's active site. The U300. A97-U277 base triple is not formed in the recently reported crystallographic model of a portion of the group I intron, despite the presence of J8/7 and P3 in the RNA construct [Golden, B. L., Gooding, A. R., Podell, E. R. & Cech, T. R. (1998) Science 282, 259-264]. This, along with other biochemical evidence, suggests that the active site in the crystallized form of the ribozyme is not fully preorganized and that substantial rearrangement may be required for substrate helix docking and catalysis.

A hydrogen-bonding triad stabilizes the chemical transition state of a group I ribozyme

The group I intron is an RNA enzyme capable of efficiently catalyzing phosphoryl-transfer reactions. Functional groups that stabilize the chemical transition state of the cleavage reaction have been identified, but they are all located within either the 5'-exon (P1) helix or the guanosine cofactor, which are the substrates of the reaction. Functional groups within the ribozyme active site are also expected to assist in transition-state stabilization, and their role must be explored to understand the chemical basis of group I intron catalysis. Using nucleotide analog interference mapping and site-specific functional group substitution experiments, we demonstrate that the 2'-OH at A207, a highly conserved nucleotide in the ribozyme active site, specifically stabilizes the chemical transition state by approximately 2 kcal mol-1. The A207 2'-OH only makes its contribution when the U(-1) 2'-OH immediately adjacent to the scissile phosphate is present, suggesting that the 2'-OHs of A207 and U(-1) interact during the chemical step. These data support a model in which the 3'-oxyanion leaving group of the transesterification reaction is stabilized by a hydrogen-bonding triad consisting of the 2'-OH groups of U(-1) and A207 and the exocyclic amine of G22. Because all three nucleotides occur within highly conserved non-canonical base pairings, this stabilization mechanism is likely to occur throughout group I introns. Although this mechanism utilizes functional groups distinctive of RNA enzymes, it is analogous to the transition states of some protein enzymes that perform similar phosphoryl-transfer reactions.

Ribozymes: the hammerhead swings into action.

A new crystal structure of a modified hammerhead ribozyme reveals an intermediate conformation that may explain discrepancies between previous structures and the required orientation of the labile bond in the ribozyme's active site.

In vitro selection for altered divalent metal specificity in the RNase P RNA.

The ribozyme RNase P absolutely requires divalent metal ions for catalytic function. Multiple Mg2+ ions contribute to the optimal catalytic efficiency of RNase P, and it is likely that the tertiary structure of the ribozyme forms a specific metal-binding pocket for these ions within the active-site. To identify base moieties that contribute to catalytic metal-binding sites, we have used in vitro selection to isolate variants of the Escherichia coli RNase P RNA with altered specificities for divalent metal. RNase P RNA variants with increased activity in Ca2+ were enriched over 18 generations of selection for catalysis in the presence of Ca2+, which is normally disfavored relative to Mg2+. Although a wide spectrum of mutations was found in the generation-18 clones, only a single point mutation was common to all clones: a cytosine-to-uracil transition at position 70 (E. coli numbering) of RNase P. Analysis of the C70U point mutant in a wild-type background confirmed that the identity of the base at position 70 is the sole determinant of Ca2+ selectivity. It is noteworthy that C70 lies within the phylogenetically well conserved J3/4-P4-J2/4 region, previously implicated in Mg2+ binding. Our finding that a single base change is sufficient to alter the metal preference of RNase P is further evidence that the J3/4-P4-J2/4 domain forms a portion of the ribozyme's active site.

Mutations in the Tetrahymena ribozyme internal guide sequence: effects on docking of the P1 helix into the catalytic core and correlation with catalytic activity.

Binding of substrate by the ribozyme derived from the self-splicing intron of Tetrahymena thermophila involves at least two steps. In the first step, base pairing between the ribozyme internal guide sequence (IGS) and the substrate forms a helical duplex (P1). Through specific tertiary interactions between P1 and the ribozyme core, P1 is then docked into the ribozyme active site. We have investigated the effects of compensatory mutations in positions 2-6 of the P1 helix on docking of P1 into the ribozyme core. Equilibrium binding of matching oligonucleotides by catalytically active IGS mutant ribozymes was evaluated by gel-shift analysis. While the strength of base pairing changed with base composition as expected, the strength of tertiary interactions between P1 and the ribozyme core was not affected by the P1 mutations. These results support a model in which efficient docking of P1 is determined by P1 structure and the presence of a conserved G-U pair. Determination of the rate of dissociation of matching oligonucleotides from each ribozyme revealed that mutations in the IGS change the tightness of binding by increasing or decreasing the dissociation rate. Surprisingly, dissociation rates determined in this fashion were 20-900-fold less than the values of the multiple-turnover rate constant for these ribozymes, initially suggesting that turnover did not require product dissociation. A more detailed analysis for the wild-type ribozyme defined two distinct product dissociation rates. The slower rate equaled that determined under the conditions used for the equilibrium binding studies. The weighted average of the two dissociation rates equaled the multiple-turnover rate constant. These results are explained by a model in which ribozyme preparations consist of two ribozyme conformers: one with tight docking of P1 and another with weaker docking of P1.

Minor Groove Recognition of the Conserved G⋅U Pair at the Tetrahymena ribozyme Reaction Site

The guanine-uracil (G.U) base pair that helps to define the 5'-splice site of group I introns is phylogenetically highly conserved. In such a wobble base pair, G makes two hydrogen bonds with U in a geometry shifted from that of a canonical Watson-Crick pair. The contribution made by individual functional groups of the G.U pair in the context of the Tetrahymena ribozyme was examined by replacement of the G.U pair with synthetic base pairs that maintain a wobble configuration, but that systematically alter functional groups in the major and minor grooves of the duplex. The substitutions demonstrate that the exocyclic amine of G, when presented on the minor groove surface by the wobble base pair conformation, contributes substantially (2 kilocalories.mole-1) to binding by making a tertiary interaction with the ribozyme active site. It contributes additionally to transition state stabilization. The ribozyme active site also makes tertiary contacts with a tripod of 2'-hydroxyls on the minor groove surface of the splice site helix. This suggests that the ribozyme binds the duplex primarily in the minor groove. The alanyl aminoacyl transfer RNA (tRNA) synthetase recognizes the exocyclic amine of an invariant G.U pair and contacts a similar array of 2'-hydroxyls when binding the tRNA(Ala) acceptor stem, providing an unanticipated parallel between protein-RNA and RNA-RNA interactions.

Characterization of the mode of binding of substrates to the active site of Tetrahymena self-splicing RNA using 5-fluorouracil-substituted mini-exons.

Splice-site selection specificity in Tetrahymena self-splicing RNA is thought to be mediated by a base-paired complex between a CUCUCU sequence on the end of the 5' exon and a GGGAGG guide sequence in the intron. The substitution of uracil (U) in oligonucleotide mini-exons with 5-fluorouracil (UF), an analogue bearing a much more acidic N-3 proton, allowed us to test the role of hydrogen bonding between complementary bases in the splice-site selection process. The affinities of (U) and (UF) mini-exons for the ribozyme active site were similar and several orders of magnitude greater than expected from base pairing alone. In contrast to CUCU, the CUFCUF mini-exon lost substrate activity with increasing pH, presumably due to ionization of the UF residues. However, the apparent pK values of these residues were several pK units above that of free UF, indicating that the mini-exon is shielded from the solvent by an active site of low polarity. Loss of the pyrimidine N-3 hydrogen bond by selective ionization of the UF residues decreased the binding of CUFCUF to the ribozyme only 3-fold but did prevent its ligation to the 3' exon. Temperature dependence of substrate activity was identical for both (U) and (UF) mini-exons, whereas the UF-substituted ribozyme lost activity at a considerably lower temperature than did the natural (U) ribozyme. These observations indicate that hydrogen-bonded base pairs involving the U residues contribute little to the total binding energy of the 5' splice site with the active site of the ribozyme, but probably help to align the splice sites properly for ligation.