Structure of the ribozyme substrate hairpin of Neurospora VS RNA: a close look at the cleavage site

The cleavage site of the Neurospora VS RNA ribozyme is located in a separate hairpin domain containing a hexanucleotide internal loop with an A-C mismatch and two adjacent G-A mismatches. The solution structure of the internal loop and helix la of the ribozyme substrate hairpin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The 2 nt in the internal loop, flanking the cleavage site, a guanine and adenine, are involved in two sheared G.A base pairs similar to the magnesium ion-binding site of the hammerhead ribozyme. Adjacent to the tandem G.A base pairs, the adenine and cytidine, which are important for cleavage, form a noncanonical wobble A+-C base pair. The dynamic properties of the internal loop and details of the high-resolution structure support the view that the hairpin structure represents a ground state, which has to undergo a conformational change prior to cleavage. Results of chemical modification and mutagenesis data of the Neurospora VS RNA ribozyme can be explained in context with the present three-dimensional structure.     

The pH dependence of hairpin ribozyme catalysis reflects ionization of an active site adenine

Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis. We developed a way to determine microscopic pK(a) values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pK(a) for ionization of 8azaA at position 38 with the apparent pK(a) for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pK(a) values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5'-oxygen-phosphorus bond.     

Role of an active site adenine in hairpin ribozyme catalysis

The hairpin ribozyme is a small catalytic RNA that accelerates reversible cleavage of a phosphodiester bond. Structural and mechanistic studies suggest that divalent metals stabilize the functional structure but do not participate directly in catalysis. Instead, two active site nucleobases, G8 and A38, appear to participate in catalytic chemistry. The features of A38 that are important for active site structure and chemistry were investigated by comparing cleavage and ligation reactions of ribozyme variants with A38 modifications. An abasic substitution of A38 reduced cleavage and ligation activity by 14,000-fold and 370,000-fold, respectively, highlighting the critical role of this nucleobase in ribozyme function. Cleavage and ligation activity of unmodified ribozymes increased with increasing pH, evidence that deprotonation of some functional group with an apparent pK(a) value near 6 is important for activity. The pH-dependent transition in activity shifted by several pH units in the basic direction when A38 was substituted with an abasic residue, or with nucleobase analogs with very high or low pK(a) values that are expected to retain the same protonation state throughout the experimental pH range. Certain exogenous nucleobases that share the amidine group of adenine restored activity to abasic ribozyme variants that lack A38. The pH dependence of chemical rescue reactions also changed according to the intrinsic basicity of the rescuing nucleobase, providing further evidence that the protonation state of the N1 position of purine analogs is important for rescue activity. These results are consistent with models of the hairpin ribozyme catalytic mechanism in which interactions with A38 provide electrostatic stabilization to the transition state.     

Solution structure of the loop B domain from the hairpin ribozyme

The hairpin ribozyme is a small catalytic RNA with a unique two-domain structure. Here we present the solution structure of the loop B domain of the hairpin ribozyme, which contains most of the catalytically essential nucleotides. The 38-nucleotide domain contains a 16-nucleotide internal loop that forms one of the largest non-Watson-Crick segments of base pairing thus far determined by either NMR or crystallography. Since the solution structure of the smaller loop A domain has been previously solved, an NMR structure-based model of the 22,000 Mr hairpin ribozyme-substrate open complex emerges by joining the two domain structures. Strikingly, catalytically essential functional groups for the loop B domain are concentrated within an expanded minor groove, presenting a clear docking surface for the loop A domain.     

Mechanistic considerations for general acid-base catalysis by RNA: revisiting the mechanism of the hairpin ribozyme

Several small ribozymes carry out self-cleavage at a specific phosphodiester bond to yield 2',3'-cyclic phosphate and 5'-hydroxyl termini. Prior mechanistic and structural studies on the HDV ribozymes led to the proposal that the pK(a) of C75 is shifted toward neutrality, making it an effective general acid. Recent mechanistic studies on the hairpin ribozyme have led to models in which protonation of G8 is required for phosphodiester cleavage, either for general acid catalysis or for electrostatic stabilization. Inspection of recent crystal structures of the hairpin ribozyme, including a complex with a vanadate transition state mimic, suggests an alternative model involving general acid-base catalysis with G8 serving as the general base and A38 as the general acid. This model is consistent with the literature on the hairpin ribozyme, including pH-rate profiles of wild-type and mutant ribozymes and solvent isotope effects. General mechanistic considerations for RNA catalysis suggest that the penalty for having general acids and bases with pK(a)s removed from neutrality is not as severe as expected. These considerations suggest that general acid-base catalysis may be a common mechanistic strategy of RNA enzymes.     

Nucleobase catalysis in the hairpin ribozyme

RNA catalysis is important in the processing and translation of RNA molecules, yet the mechanisms of catalysis are still unclear in most cases. We have studied the role of nucleobase catalysis in the hairpin ribozyme, where the scissile phosphate is juxtaposed between guanine and adenine bases. We show that a modified ribozyme in which guanine 8 has been substituted by an imidazole base is active in both cleavage and ligation, with ligation rates 10-fold faster than cleavage. The rates of both reactions exhibit bell-shaped dependence on pH, with pK(a) values of 5.7 +/- 0.1 and 7.7 +/- 0.1 for cleavage and 6.1 +/- 0.3 and 6.9 +/- 0.3 for ligation. The data provide good evidence for general acid-base catalysis by the nucleobases.     

Acid-induced exchange of the imino proton in G.C pairs.

Acid-induced catalysis of imino proton exchange in G.C pairs of DNA duplexes is surprisingly fast, being nearly as fast as for the isolated nucleoside, despite base-pair dissociation constants in the range of 10(-5) at neutral or basic pH. It is also observed in terminal G.C pairs of duplexes and in base pairs of drug-DNA complexes. We have measured imino proton exchange in deoxyguanosine and in the duplex (ATATAGATCTATAT) as a function of pH. We show that acid-induced exchange can be assigned to proton transfer from N7-protonated guanosine to cytidine in the open state of the pair. This is faster than transfer from neutral guanosine (the process of intrinsic catalysis previously characterized at neutral ph) due to the lower imino proton pK of the protonated form, 7.2 instead of 9.4. Other interpretations are excluded by a study of exchange catalysis by formiate and cytidine as exchange catalysts. The cross-over pH between the regimes of pH-independent and acid-induced exchange rates is more basic in the case of base pairs than in the mononucleoside, suggestive of an increase by one to two decades in the dissociation constant of the base pair upon N7 protonation of G. Acid-induced catalysis is much weaker in A.T base pairs, as expected in view of the low pK for protonation of thymidine.     

Catalytic roles for proton transfer and protonation in ribozymes

Utilization of proton transfer in catalysis, which is well known in the mechanisms of protein enzymes, has been described only relatively recently for RNA enzymes. In this article, we present a current understanding of proton transfer by nucleic acids. Rate enhancement and specificity conferred by general acid-base catalysis are discussed. We also present possibilities for electrostatic catalysis from general acids and bases as well as cationic base pairs. The microenvironments of a large RNA provide the possibility of histidine-like pK(a)s for proton transfer, as well as lysine- and arginine-like pK(a)s for electrostatic catalysis. Discussion on proton transfer focuses on the hepatitis delta virus (HDV) and hairpin ribozymes, with select examples drawn from the protein literature. Discussion on electrostatic catalysis also draws on these two ribozymes, and a postulate for electrostatic catalysis by a cationic base pair in the mechanism of peptidyl transfer in the ribosome is presented. We also provide a perspective on possibilities for phosphoryl transfer mechanisms involving phosphorane intermediates and unusual tautomeric forms of the bases. Lastly, a distinction is made between ground state and "transition state" pK(a)s. We favor a model in which changes in pH lead to changes in the distribution of reactive and nonreactive ionizations of the ribozyme molecules in the ground state, and therefore suggest that "pK(a) changes in the transition state" do not provide an acceptable explanation for observed pH-rate profiles.     

Ionization of a critical adenosine residue in the neurospora Varkud Satellite ribozyme active site

The Varkud Satellite (VS) ribozyme catalyzes a site-specific self-cleavage reaction that generates 5'-OH and 2',3'-cyclic phosphate products. Other ribozymes that perform an equivalent reaction appear to employ ionization of an active site residue, either to neutralize the negatively charged transition state or to act as a general acid-base catalyst. To test for important base ionization events in the VS ribozyme ligation reaction, we performed nucleotide analogue interference mapping (NAIM) with a series of ionization-sensitive adenosine and cytidine analogues. A756, a catalytically critical residue located within the VS active site, was the only nucleotide throughout the VS ribozyme that displayed the pH-dependent interference pattern characteristic of functional base ionization. We observed unique rescue of 8-azaadenosine (pK(a) 2.2) and purine riboside (pK(a) 2.1) interference at A756 at reduced reaction pH, suggestive of an ionization-specific effect. These results are consistent with protonation and/or deprotonation of A756 playing a direct role in the VS ribozyme reaction mechanism. In addition, NAIM experiments identified several functional groups within the RNA that play important roles in ribozyme folding and/or catalysis. These include residues in helix II, helix VI (730 loop), the II-III-VI and III-IV-V helix junctions, and loop V.     

Do the hairpin and VS ribozymes share a common catalytic mechanism based on general acid–base catalysis? A critical assessment of available experimental data

The active centers of the hairpin and VS ribozymes are both generated by the interaction of two internal loops, and both ribozymes use guanine and adenine nucleobases to accelerate cleavage and ligation reactions. The centers are topologically equivalent and the relative positioning of key elements the same. There is good evidence that the cleavage reaction of the VS ribozyme is catalyzed by the guanine (G638) acting as general base and the adenine (A756) as general acid. We now critically evaluate the experimental mechanistic evidence for the hairpin ribozyme. We conclude that all the available data are fully consistent with a major contribution to catalysis by general acid-base catalysis involving the adenine (A38) and guanine (G8). It appears that the two ribozymes are mechanistically equivalent.     

Comparative analysis of hairpin ribozyme structures and interference data

Great strides in understanding the molecular underpinnings of RNA catalysis have been achieved with advances in RNA structure determination by NMR spectroscopy and X-ray crystallography. Despite these successes the functional relevance of a given structure can only be assessed upon comparison with biochemical studies performed on functioning RNA molecules. The hairpin ribozyme presents an excellent case study for such a comparison. The active site is comprised of two stems each with an internal loop that forms a series of non-canonical base pairs. These loops dock into each other to create an active site for catalysis. Recently, three independent structures have been determined for this catalytic RNA, including two NMR structures of the isolated loop A and loop B stems and a high-resolution crystal structure of both loops in a docked conformation. These structures differ significantly both in their tertiary fold and the nature of the non-canonical base pairs formed within each loop. Several of the chemical groups required to achieve a functioning hairpin ribozyme have been determined by nucleotide analog interference mapping (NAIM). Here we compare the three hairpin structures with previously published NAIM data to assess the convergence between the structural and functional data. While there is significant disparity between the interference data and the individual NMR loop structures, there is almost complete congruity with the X-ray structure. The only significant differences cluster around an occluded pocket adjacent to the scissile phosphate. These local differences may suggest a role for these atoms in the transition state, either directly in chemistry or via a local structural rearrangement.     

Analysis of the functional role of a G.A sheared base pair by in vitro genetics

A classical genetic strategy has been combined with an in vitro selection method to search for functional interactions between the two domains of the hairpin ribozyme. G(21) is located within internal loop B; it is proposed to form a sheared base pair with A(43) across loop B and to bind a Mg(2+) ion. Both nucleotides are important for ribozyme function, and G.A sheared base pairs are a very widespread motif in structured RNA. We took advantage of its presence in the hairpin ribozyme to study its functional role. Pseudorevertants, in which the loss of G(21) was compensated by mutations at other positions, were isolated by in vitro selection. The vast majority of G(21) revertants contained substitutions within domain A, pointing to functional communication between specific sites within the two domains of the hairpin ribozyme. The possibility of a direct or redundant contacts is supported by electrophoretic mobility shift studies showing that a complex formed between domain B of the ribozyme and the substrate was disrupted and restored by base substitutions that have analogous effects on catalytic activity. The functional significance of this complex, the role of the nucleotides involved, and the basis for magnesium ion requirement is discussed.     

NMR structure of the A730 loop of the Neurospora VS ribozyme: insights into the formation of the active site

The Neurospora VS ribozyme is a small nucleolytic ribozyme with unique primary, secondary and global tertiary structures, which displays mechanistic similarities to the hairpin ribozyme. Here, we determined the high-resolution NMR structure of a stem-loop VI fragment containing the A730 internal loop, which forms part of the active site. In the presence of magnesium ions, the A730 loop adopts a structure that is consistent with existing biochemical data and most likely reflects its conformation in the VS ribozyme prior to docking with the cleavage site internal loop. Interestingly, the A730 loop adopts an S-turn motif that is also present in loop B within the hairpin ribozyme active site. The S-turn appears necessary to expose the Watson-Crick edge of a catalytically important residue (A756) so that it can fulfill its role in catalysis. The A730 loop and the cleavage site loop of the VS ribozyme display structural similarities to internal loops found in the active site of the hairpin ribozyme. These similarities provided a rationale to build a model of the VS ribozyme active site based on the crystal structure of the hairpin ribozyme.     

A guanine nucleobase important for catalysis by the VS ribozyme

A guanine (G638) within the substrate loop of the VS ribozyme plays a critical role in the cleavage reaction. Replacement by any other nucleotide results in severe impairment of cleavage, yet folding of the substrate is not perturbed, and the variant substrates bind the ribozyme with similar affinity, acting as competitive inhibitors. Functional group substitution shows that the imino proton on the N1 is critical, suggesting a possible role in general acid-base catalysis, and this in accord with the pH dependence of the reaction rate for the natural and modified substrates. We propose a chemical mechanism for the ribozyme that involves general acid-base catalysis by the combination of the nucleobases of guanine 638 and adenine 756. This is closely similar to the probable mechanism of the hairpin ribozyme, and the active site arrangements for the two ribozymes appear topologically equivalent. This has probably arisen by convergent evolution.     

Solution structure of a DNA double helix incorporating four consecutive non-Watson-Crick base-pairs

A series of DNA 21-mers containing a variety of the 4 x 4 internal loop sequence 5'-CAAG-3'/3'-ACGT-5' were studied using nuclear magnetic resonance (NMR) methodology and distance geometry (DG)/molecular dynamics (MD) approaches. Such oligomers exhibit excellent resolution in the NMR spectra and reveal many unusual NOEs (nuclear Overhauser effect) that allow for the detailed characterization of a DNA hairpin incorporating a track of four different non-Watson-Crick base-pairs in the stem. These include a wobble C.A base-pair, a sheared A.C base-pair, a sheared A.G base-pair, and a wobble G.T base-pair. Significantly different twisting angles were observed between the base-pairs in internal loop that results with excellent intra-strand and inter-strand base stacking within the four consecutive mismatches and the surrounding canonical base-pairs. This explains why it melts at 52 degrees C even though five out of ten base-pairs in the stem adopt non-Watson-Crick pairs. However, the 4 x 4 internal loop still fits into a B-DNA double helix very well without significant change in the backbone torsion angles; only zeta torsion angles between the tandem sheared base-pairs are changed to a great extent from the gauche(-) domain to the trans domain to accommodate the cross-strand base stacking in the internal loop. The observation that several consecutive non-canonical base-pairs can stably co-exist with Watson-Crick base-pairs greatly increases the limited repertoire of irregular DNA folds and reveals the possibility for unusual structural formation in the functionally important genomic regions that have potential to become single-stranded.     

Role of an active site guanine in hairpin ribozyme catalysis probed by exogenous nucleobase rescue

The hairpin ribozyme is a small catalytic RNA with reversible phosphodiester cleavage activity. Biochemical and structural studies exclude a requirement for divalent metal cation cofactors and implicate one active site nucleobase in particular, G8, in the catalytic mechanism. Our previous work demonstrated that the cleavage activity that is lost when G8 is replaced by an abasic residue is restored when certain nucleobases are provided in solution. The specificity and pH dependence of exogenous nucleobase rescue were consistent with several models of the rescue mechanism, including general acid base catalysis, electrostatic stabilization of negative charge in the transition state or a requirement for protonation to facilitate exogenous nucleobase binding. Detailed analyses of exogenous nucleobase rescue for both cleavage and ligation reactions now allow us to refine models of the rescue mechanism. Activity increased with increasing pH for both unmodified ribozyme reactions and unrescued reactions of abasic variants lacking G8. This similarity in pH dependence argues against a role for G8 as a general base catalyst, because G8 deprotonation could not be responsible for the pH-dependent transition in the abasic variant. Exogenous nucleobase rescue of both cleavage and ligation activity increased with decreasing pH, arguing against a role for rescuing nucleobases in general acid catalysis, because a nucleobase that contributes general acid catalysis in the cleavage pathway should provide general base catalysis in ligation. Analysis of the concentration dependence of cytosine rescue at high and low pH demonstrated that protonation promotes catalysis within the nucleobase-bound ribozyme complex but does not stabilize nucleobase binding in the ground state. These results support an electrostatic stabilization mechanism in which exogenous nucleobase binding counters negative charge that develops in the transition state.     

Catalysis by the nucleolytic ribozymes

The nucleolytic ribozymes use general acid-base catalysis to contribute significantly to their rate enhancement. The VS (Varkud satellite) ribozyme uses a guanine and an adenine nucleobase as general base and acid respectively in the cleavage reaction. The hairpin ribozyme is probably closely similar, while the remaining nucleolytic ribozymes provide some interesting contrasts.     

Protonation of deoxycytidine residues in dC4 tetraloops: UV spectrophotometric study of dC10 and d(A14C4T14)

It is shown that component analysis could be applied to study the UV difference spectra of cytidine oligomers and hairpin oligonucleotides with cytidines in the loop region in order to account for the melting and titration results in terms of cytidine stacking and protonation. Upon acid titration, the dC(10) oligomer undergoes cooperative conformational transition at pH 6.3 accompanied by protonation and formation of the i-structure with half of the residues protonated. The stability of the hemiprotonated structure increases with decreasing pH, the i-structure persisting still in the region of pH相似文献   

pK(a) calculations along a bacteriorhodopsin molecular dynamics trajectory

Electrostatic calculations of pK(a-values) are reported along a 400 ps molecular dynamics trajectory of bacteriorhodopsin. The sensitivity of calculated pK(a) values to a number of structural factors and factors related to the modelling of the electrostatics are also studied. The results are very sensitive to the choice of internal dielectric constant of the protein (in the interval 2-4). Moreover it is important to include internal water molecules and to average over a long enough portion ( approximately 100 ps) of an equilibrium molecular dynamics trajectory. The internal waters are necessary to get an ion-counter ion complex with the Schiff base and Arg 82 protonated and the aspartic groups (85 and 212) deprotonated. The fluctuations along the MD-trajectory do not change the protonation state of internal residues at neutral pH. However, at other pH values the averaging along a trajectory maybe crucial to get correct protonation states. A relationship is found between the arginine group 82, the aspartic group 85 and the glutamate group 204. Glu 204 is protonated in the ground state but the pK(a) value decreases towards deprotonation when the chromophore isomerizes into the cis state.     

pK(a) calculations for class C beta-lactamases: the role of Tyr-150

The Poisson-Boltzmann method was used to compute the pK(a) values of titratable residues in a set of class C beta-lactamases. In these calculations, the pK(a) of the phenolic group of residue Tyr150 is the only one to stand out with an abnormally low value of 8.3, more than one pK(a) unit lower than the measured reference value for tyrosine in solution. Other important residues of the catalytic pocket, such as the conserved Lys67, Lys315, His314, and Glu272 (hydrogen-bonded to the ammonium group of Lys315), display normal protonation states at neutral pH. pK(a) values were also computed in catalytically impaired beta-lactamase mutants. Comparisons between the relative k(cat) values and the Tyr150 pK(a) value in these mutants revealed a striking correlation. In active enzymes, this pK(a) value is always lower than the solution reference value while it is close to normal in inactive enzymes. These results thus support the hypothesis that the phenolate form of Tyr150 is responsible for the activation of the nucleophilic serine. The possible roles of Lys67 and Lys315 during catalysis are also discussed.