Structure and function of the hairpin ribozyme

The hairpin ribozyme belongs to the family of small catalytic RNAs that cleave RNA substrates in a reversible reaction that generates 2',3'-cyclic phosphate and 5'-hydroxyl termini. The hairpin catalytic motif was discovered in the negative strand of the tobacco ringspot virus satellite RNA, where hairpin ribozyme-mediated self-cleavage and ligation reactions participate in processing RNA replication intermediates. The self-cleaving hairpin, hammerhead, hepatitis delta and Neurospora VS RNAs each adopt unique structures and exploit distinct kinetic and catalytic mechanisms despite catalyzing the same chemical reactions. Mechanistic studies of hairpin ribozyme reactions provided early evidence that, like protein enzymes, RNA enzymes are able to exploit a variety of catalytic strategies. In contrast to the hammerhead and Tetrahymena ribozyme reactions, hairpin-mediated cleavage and ligation proceed through a catalytic mechanism that does not require direct coordination of metal cations to phosphate or water oxygens. The hairpin ribozyme is a better ligase than it is a nuclease while the hammerhead reaction favors cleavage over ligation of bound products by nearly 200-fold. Recent structure-function studies have begun to yield insights into the molecular bases of these unique features of the hairpin ribozyme.     

Evidence for a hydroxide ion bridging two magnesium ions at the active site of the hammerhead ribozyme.

In the presence of magnesium ions, cleavage by the hammerhead ribozyme RNA at a specific residue leads to 2'3'-cyclic phosphate and 5'-OH extremities. In the cleavage reaction an activated ribose 2'-hydroxyl group attacks its attached 3'-phosphate. Molecular dynamics simulations of the crystal structure of the hammerhead ribozyme, obtained after flash-freezing of crystals under conditions where the ribozyme is active, provide evidence that a mu-bridging OH-ion is located between two Mg2+ions close to the cleavable phosphate. Constrained simulations show further that a flip from the C3'- endo to the C2'- endo conformation of the ribose at the cleavable phosphate brings the 2'-hydroxyl in proximity to both the attacked phosphorous atom and the mu-bridging OH-ion. Thus, the simulations lead to a detailed new insight into the mechanism of hammerhead ribozyme cleavage where a mu-hydroxo bridged magnesium cluster, located on the deep groove side, provides an OH-ion that is able to activate the 2'-hydroxyl nucleophile after a minor and localized conformational change in the RNA.     

Specificity from steric restrictions in the guanosine binding pocket of a group I ribozyme

The 3' splice site of group I introns is defined, in part, by base pairs between the intron core and residues just upstream of the splice site, referred to as P9.0. We have studied the specificity imparted by P9.0 using the well-characterized L-21 Scal ribozyme from Tetrahymena by adding residues to the 5' end of the guanosine (G) that functions as a nucleophile in the oligonucleotide cleavage reaction: CCCUCUA5 (S) + NNG <--> CCCUCU + NNGA5. UCG, predicted to form two base pairs in P9.0, reacts with a (kcat/KM) value approximately 10-fold greater than G, consistent with previous results. Altering the bases that form P9.0 in both the trinucleotide G analog and the ribozyme affects the specificity in the manner predicted for base-pairing. Strikingly, oligonucleotides incapable of forming P9.0 react approximately 10-fold more slowly than G, for which the mispaired residues are simply absent. The observed specificity is consistent with a model in which the P9.0 site is sterically restricted such that an energetic penalty, not present for G, must be overcome by G analogs with 5' extensions. Shortening S to include only one residue 3' of the cleavage site (CCCUCUA) eliminates this penalty and uniformly enhances the reactions of matched and mismatched oligonucleotides relative to guanosine. These results suggest that the 3' portion of S occupies the P9.0 site, sterically interfering with binding of G analogs with 5' extensions. Similar steric effects may more generally allow structured RNAs to avoid formation of incorrect contacts, thereby helping to avoid kinetic traps during folding and enhancing cooperative formation of the correct structure.     

The internal equilibrium of the hairpin ribozyme: temperature, ion and pH effects

The hairpin ribozyme reversibly cleaves phosphodiesters of RNA substrates to generate products with 5' hydroxyl and 2',3'-cyclic phosphate termini. We previously found that the rate constant for ligation is tenfold faster than the rate constant for cleavage under standard conditions. The hammerhead ribozyme catalyzes the same reactions but is reported to favor cleavage relative to ligation by more than 100-fold under the same conditions. To explore the basis for this difference, we examined the influence of temperature, ions and pH on the hairpin ribozyme internal equilibrium. Under the same conditions, the loss of entropy associated with ligation is less for the hairpin than for the hammerhead ribozyme, consistent with the notion that a more rigid hairpin structure undergoes a smaller decrease in dynamics upon ligation than the more flexible hammerhead structure. Increased salt and reduced temperature shift the equilibrium toward ligation while pH has little effect, suggesting that conditions that stabilize RNA structure tend to promote ligation. The hairpin ribozyme appears to take up at least one tri- or divalent cation or two monovalent cations upon ligation. The efficiency with which different cations promote ligation depends strongly on valence and, less strongly, on ionic radius or electronegativity. This pattern of cation selectivity suggests that cations promote ligation through delocalized electrostatic shielding, perhaps interacting with a region of especially high charge density in the ligated ribozyme. Changes in ionic conditions produce large but compensating changes in enthalpy and entropy for cleavage and ligation. Thus, in addition to any increase in ribozyme dynamics associated with cleavage, re-organization of associated cations contributes significantly to hairpin ribozyme thermodynamics.     

Importance of short pseudoknot base pairs between two single-stranded regions of HDV ribozyme.

Human hepatitis delta virus (HDV) ribozyme can catalyze self-cleavage reaction in the presence of Mg2+ ions, yielding products with 2',3'-cyclic phosphate and 5'-OH termini as do hammerhead and hairpin ribozymes. Recently, the tertiary structure of 3'-cleaved product of genomic HDV ribozyme was solved by X-ray crystallographic analysis. In this structure three single-stranded regions (SSrA, -B and -C) interacts intricately with hydrogen bonds between bases, phosphate oxygens and 2'-OHs to form nested double pseudoknot structure. Especially two Watson-Crick base pairs, 726G-710C and 727G-709C, between SSrA and SSrC, seems to be important for compact folding. To characterize the necessity of the two base pairs, we performed in vitro selection of active ribozymes using random RNA pool which mutated at 709, 710, 726 and 727. The result indicates that basically one G-C base pair is necessary for the activity.     

Capture and visualization of a catalytic RNA enzyme-product complex using crystal lattice trapping and X-ray holographic reconstruction

We have determined the crystal structure of the enzyme-product complex of the hammerhead ribozyme by using a reinforced crystal lattice to trap the complex prior to dissociation and by employing X-ray holographic image reconstruction, a real-space electron density imaging and refinement procedure. Subsequent to catalysis, the cleavage site residue (C-17), together with its 2',3'-cyclic phosphate, adopts a conformation close to and approximately perpendicular to the Watson-Crick base-pairing faces of two highly conserved purines in the ribozyme's catalytic pocket (G-5 and A-6). We observe several interactions with functional groups on these residues that have been identified as critical for ribozyme activity by biochemical analyses but whose role has defied explanation in terms of previous structural analyses. These interactions may therefore be relevant to the hammerhead ribozyme reaction mechanism.     

Regulation of beta1 integrin expression in endothelial cells by chimeric tRNA(Val) ribozyme

To downregulate expression of the beta1 integrin subunit in endothelial cells, plasmid encoding the ribozyme cassette containing hammerhead ribozyme flanked at the 5' terminus by tRNA(Val) and at the 3' terminus by constitutive transport element sequences was constructed. When used to transfect immortalized human endothelial cell line EA.hy 926, it selectively blocked the synthesis of the beta1 integrin subunit and thus inhibited migration and proliferation of the cells. Thus, this construct may be a valuable tool to control the proangiogenic phenotype of stimulated endothelial cells.     

The hairpin ribozyme

The hairpin ribozyme is a member of a family of small RNA endonucleases, which includes hammer-head, human hepatitis delta virus, Neurospora VS, and the lead-dependent catalytic RNAs. All these catalytic RNAs reversibly cleave the phosphodiester bond of substrate RNA to generate 5'-hydroxyl and 2',3'-cyclic phosphate termini. Whereas the reaction products from family members are similar, large structural and mechanistic differences exist. Structurally the hairpin ribozyme has two principal domains that interact to facilitate catalysis. The hairpin ribozyme uses a catalytic mechanism that does not require metals for cleavage or ligation of substrate RNA. In this regard it is presently unique among RNA catalysts. Targeting rules for cleavage of substrate have been determined and required bases for catalysis have been identified. The hairpin ribozyme has been developed and used for gene therapy and was the first ribozyme to be approved for human clinical trials.     

Molecular dynamics investigations of hammerhead ribozyme RNA

The hammerhead ribozyme, a small catalytic RNA molecule, cleaves, in the presence of magnesium ions, a specific phosphodiester bond within its own backbone, leading to 23-cyclic phosphate and 5-OH extremities. In order to study the dynamical flexibility of the hammerhead RNA, we performed molecular dynamics simulations of the solvated crystal structure of an active hammerhead ribozyme, obtained after flash-freezing crystals soaked with magnesium. Because of a careful equilibration protocol and the use of the Ewald summation in calculating the electrostatic interactions, the RNA structure remained close to the crystal structure, as attested by a root-mean-square deviation below 2.5 A after 750 ps of simulation. All Watson-Crick base pairs were intact at the end of the simulations. The tertiary interactions, such as the sheared G.A pairs and the U-turn, important for the stabilisation of the three-dimensional RNA fold, were also retained. The results demonstrate that molecular dynamics simulations can be successfully used to investigate the dynamical behaviour of a ribozyme, thus, opening a road to study the role of transient structural changes involved in ribozyme catalysis.     

Specificity of hammerhead ribozyme cleavage.

To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths.     

In vitro evolution of the hammerhead ribozyme to a purine-specific ribozyme using mutagenic PCR with two nucleotide analogues

The conventional hammerhead ribozyme cleaves RNA 3' to nucleotide triplets with the general formula NUH, where N is any nucleotide, U is uridine and H is any nucleotide except guanosine. In order to isolate hammerhead ribozyme sequences capable of cleaving 3' to the GUG triplet, we performed a mutagenic selection protocol starting with the conventional sequence of an NUH-cleaving ribozyme. The 22 nucleotides in the core and the stem-loop II region were subjected to mutagenic PCR using the two nucleotide analogues 6-(2-deoxy-beta-d-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C)][1, 2] oxazin-7-one and of 8-oxo-2'-deoxyguanosine. After five repetitions of the selection cycle, several clones showed cleavage activity. One sequence, having one deletion, showed at least a 90 times higher in trans cleavage rate than the starting ribozyme. It cleaved 3' to GUG and GUA. The sequence of this ribozyme is essentially identical with that obtained previously by selection for AUG cleavage starting with a randomised core and stem-loop II region. This identical result of two independent selection procedures supports the notion that sequences for NUR cleavage, where R is a purine nucleotide, are not compatible with the classical hammerhead structure, and that the sequence space for this cleavage specificity is very limited. The cleavage of NUR triplets is not restricted to the sequence of the substrate that was used for selection but is sequence-independent for in trans cleavage, although the sequence context influences the value for the cleavage rate somewhat. Analysis of cleavage activities indicates the importance of A at position L2.5 in loop II.     

Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic description of the reaction of an RNA substrate that forms a mismatch at the active site

The site-specific endonuclease reaction catalyzed by the ribozyme from the Tetrahymena pre-rRNA intervening sequence has been characterized with a substrate that forms a "matched" duplex with the 5' exon binding site of the ribozyme [G2CCCUCUA5 + G in equilibrium with G2CCCUCU + GA5 (G = guanosine); Herschlag, D., & Cech, T.R. (1990) Biochemistry (preceding paper in this issue)]. The rate-limiting step with saturating substrate is dissociation of the product G2CCCUCU. Here we show that the reaction of the substrate G2CCCGCUA5, which forms a "mismatched" duplex with the 5' exon binding site at position -3 from the cleavage site, has a value of kcat that is approximately 10(2)-fold greater than kcat for the matched substrate (50 degrees C, 10 mM MgCl2, pH 7). This is explained by the faster dissociation of the mismatched product, G2CCCGCU, than the matched product. With subsaturating oligonucleotide substrate and saturating G, the binding of the oligonucleotide substrate and the chemical step are each partially rate-limiting. The rate constant for the chemical step of the endonuclease reaction and the rate constant for the site-specific hydrolysis reaction, in which solvent replaces G, are each within approximately 2-fold with the matched and mismatched substrates, despite the approximately 10(3)-fold weaker binding of the mismatched substrate. This can be described as "uniform binding" of the base at position -3 in the ground state and transition state [Albery, W.J., & Knowles, J. R. (1976) Biochemistry 15, 5631-5640]. Thus, the matched substrate does not use its extra binding energy to preferentially stabilize the transition state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ribozyme cleavage of a 2,5-phosphodiester linkage: mechanism and a restricted divalent metal-ion requirement.

The natural substrate cleaved by the hepatitis delta virus (HDV) ribozyme contains a 3',5'-phosphodiester linkage at the cleavage site; however, a 2',5'-linked ribose-phosphate backbone can also be cleaved by both trans-acting and self-cleaving forms of the HDV ribozyme. With substrates containing either linkage, the HDV ribozyme generated 2',3'-cyclic phosphate and 5'-hydroxyl groups suggesting that the mechanisms of cleavage in both cases were by a nucleophilic attack on the phosphorus center by the adjacent hydroxyl group. Divalent metal ion was required for cleavage of either linkage. However, although the 3',5'-linkage was cleaved slightly faster in Ca2+ than in Mg2+, the 2',5'-linkage was cleaved in Mg2+ (or Mn2+) but not Ca2+. This dramatic difference in metal-ion specificity is strongly suggestive of a crucial metal-ion interaction at the active site. In contrast to the HDV ribozymes, cleavage at a 2',5'-phosphodiester bond was not efficiently catalyzed by the hammerhead ribozyme. The relaxed linkage specificity of the HDV ribozymes may be due in part to lack of a rigid binding site for sequences 5' to the cleavage site.     

Inhibition of hepatitis B virus by hammerhead ribozyme targeted to the poly(A) signal sequence in cultured cells

The deviant poly(A) signal of hepatitis B virus (HBV) not only controls the formation of the 3' end of all the viral RNA, but is also crucial for HBV replication. Hence, a cis-releasing hammerhead ribozyme (RzA) targeted to the poly(A) signal region of HBV subtype adr was investigated for its antiviral effects. In vitro, RzA cleaved HBV RNA at its target site up to 70%, while the disabled ribozyme (dRzA), which had a one-base mutation in the catalytic site, did not cleave the target RNA at all. When the ribozymes were cotransfected into HepG2 cells with the HBV genome-containing plasmid p3.6II, the wild-type ribozyme RzA could effectively decrease HBV RNA levels and inhibit HBV replication, whereas its disabled form, dRzA, had much weaker effects, indicating that the active catalytic domain of the hammerhead ribozyme could markedly increase the extent of antisense-mediated inhibition. In addition, there was a gradient of effectiveness: the higher the amount of released ribozyme, the more the reduction in target HBV RNA in cells as well as progeny DNA reduction. These results suggest the possibility of the hammerhead ribozyme RzA to be used for the gene therapy of HBV infection.     

The 2 A structure of helix 6 of the human signal recognition particle RNA

BACKGROUND: The mammalian signal recognition particle (SRP) is an essential cytoplasmic ribonucleoprotein complex involved in targeting signal-peptide-containing proteins to the endoplasmic reticulum. Assembly of the SRP requires protein SRP19 to bind first to helix 6 of the SRP RNA before the signal-peptide-recognizing protein, SRP54, can bind to helix 8 of the RNA. Helix 6 is closed by a GGAG tetraloop, which has been shown to form part of the SRP19-binding site. RESULTS: The high-resolution (2.0 A) structure of a fragment of human SRP RNA comprising 29 nucleotides of helix 6 has been determined using the multiple anomalous dispersion (MAD) method and bromine-labelled RNA. In the crystal the molecule forms 28-mer duplexes rather than the native monomeric hairpin structure, although two chemically equivalent 11 base pair stretches of the duplex represent the presumed native structure. The duplex has highly distorted A-RNA geometry caused by the occurrence of several non-Watson-Crick base pairs. These include a 5'-GGAG-3'/3'-GAGG-5' purine bulge (which replaces the tetraloop) and a 5'-AC-3'/3'-CA-5' tandem mismatch that, depending on the protonation state of the adenine bases, adopts a different conformation in the two native-like parts of the structure. The structure also shows the 2'3'-cyclic phosphate reaction product of the hammerhead ribozyme cleavage reaction. CONCLUSIONS: The 29-mer RNA is the first RNA structure of the human SRP and provides some insight into the binding mode of SRP19. The observed strong irregularities of the RNA helix make the major groove wide enough and flat enough to possibly accommodate an alpha helix of SRP19. The variety of non-canonical base pairs observed enlarges the limited repertoire of irregular RNA folds known to date and the observed conformation of the 2'3'-cyclic phosphate containing Ade29 is consistent with the current understanding of the hammerhead ribozyme reaction mechanism.     

Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids.     

Tertiary contacts distant from the active site prime a ribozyme for catalysis

Minimal hammerhead ribozymes have been characterized extensively by static and time-resolved crystallography as well as numerous biochemical analyses, leading to mutually contradictory mechanistic explanations for catalysis. We present the 2.2 A resolution crystal structure of a full-length Schistosoma mansoni hammerhead ribozyme that permits us to explain the structural basis for its 1000-fold catalytic enhancement. The full-length hammerhead structure reveals how tertiary interactions occurring remotely from the active site prime this ribozyme for catalysis. G-12 and G-8 are positioned consistent with their previously suggested roles in acid-base catalysis, the nucleophile is aligned with a scissile phosphate positioned proximal to the A-9 phosphate, and previously unexplained roles of other conserved nucleotides become apparent within the context of a distinctly new fold that nonetheless accommodates the previous structural studies. These interactions permit us to explain the previously irreconcilable sets of experimental results in a unified, consistent, and unambiguous manner.