Effects of cobalt hexammine on folding and self-cleavage of the Neurospora VS ribozyme

We have investigated the effects of Co(NH3)6(3+), an analog of hexahydrated Mg2+, on folding and catalysis of the Neurospora VS ribozyme. Most of the metal ion-induced changes detected by chemical modification structure probing in either metal ion are similar, but occur at approximately 33-fold lower concentrations of Co(NH3)6(3+) than Mg2+. However, Co(NH3)6(3+) is not as effective at inducing two functionally important structural changes: stabilizing the pseudoknot interaction between loops I and V, and rearranging the secondary structure of helix Ib. Comparison of the folding of the precursor and the downstream cleavage product, which lacks helix Ia, shows that helix Ia inhibits stable pseudoknot formation and rearrangement of helix Ib. The VS ribozyme does not self-cleave with Co(NH3)6(3+) as the sole polyvalent cation; however, mixed-metal kinetic experiments show that Co(NH3)6(3+) does not inhibit Mg2+-induced self-cleavage. In contrast, at sub-saturating concentrations of Mg2+, Co(NH3)6(3+) increases the rate of Mg2+-induced self-cleavage, indicating that Co(NH3)6(3+) contributes to the functionally relevant folding of the VS ribozyme.     

Studies on Escherichia coli RNase P RNA with Zn2+ as the catalytic cofactor

We demonstrate, for the first time, catalysis by Escherichia coli ribonuclease P (RNase P) RNA with Zn2+ as the sole divalent metal ion cofactor in the presence of ammonium, but not sodium or potassium salts. Hill analysis suggests a role for two or more Zn2+ ions in catalysis. Whereas Zn2+ destabilizes substrate ground state binding to an extent that precludes reliable Kd determination, Co(NH3)6(3+) and Sr2+ in particular, both unable to support catalysis by themselves, promote high-substrate affinity. Zn2+ and Co(NH3)6(3+) substantially reduce the fraction of precursor tRNA molecules capable of binding to RNase P RNA. Stimulating and inhibitory effects of Sr2+ on the ribozyme reaction with Zn2+ as cofactor could be rationalized by a model involving two Sr2+ ions (or two classes of Sr2+ ions). Both ions improve substrate affinity in a cooperative manner, but one of the two inhibits substrate conversion in a non-competitive mode with respect to the substrate and the Zn2+. A single 2'-fluoro modification at nt -1 of the substrate substantially weakened the inhibitory effect of Sr2+. Our results demonstrate that the studies on RNase P RNA with metal cofactors other than Mg2+ entail complex effects on structural equilibria of ribozyme and substrate RNAs as well as E*S formation apart from the catalytic performance.     

Unusual metal specificity and structure of the group I ribozyme from Chlamydomonas reinhardtii 23S rRNA

Group I intron ribozymes require cations for folding and catalysis, and the current literature indicates that a number of cations can promote folding, but only Mg2+ and Mn2+ support both processes. However, some group I introns are active only with Mg2+, e.g. three of the five group I introns in Chlamydomonas reinhardtii. We have investigated one of these ribozymes, an intron from the 23S LSU rRNA gene of Chlamydomonas reinhardtii (Cr.LSU), by determining if the inhibition by Mn2+ involves catalysis, folding, or both. Kinetic analysis of guanosine-dependent cleavage by a Cr.LSU ribozyme, 23S.5 Delta Gb, that lacks the 3' exon and intron-terminal G shows that Mn2+ does not affect guanosine binding or catalysis, but instead promotes misfolding of the ribozyme. Surprisingly, ribozyme misfolding induced by Mn2+ is highly cooperative, with a Hill coefficient larger than that of native folding induced by Mg2+. At lower Mn2+ concentrations, metal inhibition is largely alleviated by the guanosine cosubstrate (GMP). The concentration dependence of guanosine cosubstrate-induced folding suggests that it functions by interacting with the G binding site, perhaps by displacing an inhibitory Mn2+. Because of these and other properties of Cr.LSU, the tertiary structure of the intron from 23S.5 Delta Gb was examined using Fe2+-EDTA cleavage. The ground-state structure shows evidence of an unusually open ribozyme core: the catalytic P3-P7 domain and the nucleotides that connect it to the P4-P5-P6 domain are exposed to solvent. The implications of this structure for the in vitro and in vivo properties of this intron ribozyme are discussed.     

Role of counterion condensation in folding of the Tetrahymena ribozyme. II. Counterion-dependence of folding kinetics

Condensed counterions contribute to the stability of compact structures in RNA, largely by reducing electrostatic repulsion among phosphate groups. Varieties of cations induce a collapsed state in the Tetrahymena ribozyme that is readily transformed to the catalytically active structure in the presence of Mg2+. Native gel electrophoresis was used to compare the effects of the valence and size of the counterion on the kinetics of this transition. The rate of folding was found to decrease with the charge of the counterion. Transitions in monovalent ions occur 20- to 40-fold faster than transitions induced by multivalent metal ions. These results suggest that multivalent cations yield stable compact structures, which are slower to reorganize to the native conformation than those induced by monovalent ions. The folding kinetics are 12-fold faster in the presence of spermidine3+ than [Co(NH3)6]3+, consistent with less effective stabilization of long-range RNA interactions by polyamines. Under most conditions, the observed folding rate decreases with increasing counterion concentration. In saturating amounts of counterion, folding is accelerated by addition of urea. These observations indicate that reorganization of compact intermediates involves partial unfolding of the RNA. We find that folding of the ribozyme is most efficient in a mixture of monovalent salt and Mg2+. This is attributed to competition among counterions for binding to the RNA. The counterion dependence of the folding kinetics is discussed in terms of the ability of condensed ions to stabilize compact structures in RNA.     

Mechanistic characterization of the HDV genomic ribozyme: classifying the catalytic and structural metal ion sites within a multichannel reaction mechanism

Prior studies of the metal ion dependence of the self-cleavage reaction of the HDV genomic ribozyme led to a mechanistic framework in which the ribozyme can self-cleave by multiple Mg2+ ion-independent and -dependent channels [Nakano et al. (2001) Biochemistry 40, 12022]. In particular, channel 2 involves cleavage in the presence of a structural Mg2+ ion without participation of a catalytic divalent metal ion, while channel 3 involves both structural and catalytic Mg2+ ions. In the present study, experiments were performed to probe the nature of the various divalent ion sites and any specificity for Mg2+. A series of alkaline earth metal ions was tested for the ability to catalyze self-cleavage of the ribozyme under conditions that favor either channel 2 or channel 3. Under conditions that populate primarily channel 3, nearly identical K(d)s were obtained for Mg2+, Ca2+, Ba2+, and Sr2+, with a slight discrimination against Ca2+. In contrast, under conditions that populate primarily channel 2, tighter binding was observed as ion size decreases. Moreover, [Co(NH3)6]3+ was found to be a strong competitive inhibitor of Mg2+ for channel 3 but not for channel 2. The thermal unfolding of the cleaved ribozyme was also examined, and two transitions were found. Urea-dependent studies gave m-values that allowed the lower temperature transition to be assigned to tertiary structure unfolding. The effects of high concentrations of Na+ on the melting temperature for RNA unfolding and the reaction rate revealed ion binding to the folded RNA, with significant competition of Na+ (Hill coefficient of 1.5-1.7) for a structural Mg2+ ion and an unusually high intrinsic affinity of the structural ion for the RNA. Taken together, these data support the existence of two different classes of metal ion sites on the ribozyme: a structural site that is inner sphere with a major electrostatic component and a preference for Mg2+, and a weak catalytic site that is outer sphere with little preference for a particular divalent ion.     

Ribozyme-catalyzed dipeptide synthesis in monovalent metal ions alone

Previously we have identified a highly active ribozyme (R180, cis ribozyme) that can catalyze dipeptide synthesis using N-biotinylcaproyl-aminoacyl-adenylate anhydride (Bio-aa-5'-AMP) as its substrate. In this work, we re-engineered the cis R180 ribozyme into a 158-nt trans ribozyme (TR158) and designed a new substrate (5'-Phe-linker-20-mer). First, the metal ion requirements were examined and compared between the two ribozymes. Both R180 and TR158 ribozymes were active in Mg2+ and Ca2+ but inert with Zn2+, Cu2+, Mn2+, and Co2+. It is intriguing that both ribozymes were highly active in Li+, Na+, or K+ alone but showed very low activity with NH4+. The two ribozymes showed similar linear concentration dependence on Li+ and K+, while they displayed different dependency behavior on Mg2+. Moreover, by using the trans system, the detailed kinetic studies and pH dependent experiments were performed in either 10 mM Mg2+ or 1.0 M Li+. Analysis of kcat and Km values obtained at different pHs (6.0 to 9.0) indicated that it is the catalytic activity of the ribozyme but not the substrate binding affinity that changes significantly with pH. The slopes of the linear parts of the pH-rate plots were close to 1.0 in both Mg2+- and Li+-mediated reactions, suggesting that one proton transfer is involved in the rate-limiting step of catalysis. Overall, our results suggest that Mg2+ and Li+ function similarly in the ribozyme-catalyzed dipeptide synthesis.     

Monovalent ion-mediated folding of the Tetrahymena thermophila ribozyme

The time-course of monovalent cation-induced folding of the L-21 Sca1 Tetrahymena thermophila ribozyme and a selected mutant was quantitatively followed using synchrotron X-ray (.OH) footprinting. Initiating folding by increasing the concentration of either Na+ or K+ to 1.5M from an initial condition of approximately 0.008 M Na+ at 42 degrees C resulted in the complete formation of tertiary contacts within the P5abc subdomain and between the peripheral helices within the dead time of our measurements (k>50 s(-1)). These results contrast with folding rates of 2-0.2 s(-1) previously observed for formation of these contacts in 10mM Mg2+ from the same initial condition. Thus, the initial formation of native tertiary contacts is inhibited by divalent but not monovalent cations. The native contacts within the catalytic core form without a detectable burst phase at rates of 0.4-1.0 s(-1) in a manner reminiscent of the Mg2+-dependent folding behavior, although tenfold faster. The tertiary interactions stabilizing the catalytic core interaction with P4-P6 and P2.1, as well as one of the protections internal for the P4-P6 domain, display progress curves with appreciable burst amplitudes and a phase comparable in rate to that of the catalytic core. That the slow folding of the ribozyme's core is a consequence of the alt-P3 secondary structure is shown by the 100% burst phase amplitudes that are observed for folding of the U273A mutant ribozyme within which the native secondary structure (P3) is strengthened. Thus, formation of a misfolded intermediate(s) resulting from the alt-P3 secondary structure is independent of ion valency while the rate at which the respective intermediates are resolved is sensitive to ion valency. The overall portrait painted by these results is that ion valency differentially affects steps in the folding process and that folding in monovalent ion alone for the U273A mutant Tetrahymena ribozyme is fast and direct.     

Perturbation of the hierarchical folding of a large RNA by the destabilization of its Scaffold's tertiary structure

The P4-P6 domain serves as a scaffold against which the periphery and catalytic core organize and fold during Mg2+-mediated folding of the Tetrahymena thermophila ribozyme. The most prominent structural motif of the P4-P6 domain is the tetraloop-tetraloop receptor interaction which "clamps" the distal parts of its hairpin-like structure. Destabilization of the tertiary structure of the P4-P6 domain by perturbation of the tetraloop-tetraloop receptor interaction alters the Mg2+-mediated folding pathway. The folding hierarchy of P5c approximately P4-P6 > periphery > catalytic core that is a striking attribute of the folding of the wild-type RNA is abolished. The initial steps in folding of the mutant RNA are > or =50-fold faster than those of the wild-type ribozyme with the earliest observed tertiary contacts forming around regions known to specifically bind Mg2+. The interaction between the mutant tetraloop and the tetraloop receptor appears coincidently with slowly forming catalytic core tertiary contacts. Thus, the stability conferred upon the P4-P6 domain by the tetraloop-tetraloop receptor interaction dictates the preferred folding pathway by stabilizing an early intermediate. A sub-denaturing concentration of urea diminishes the early barrier to folding the wild-type ribozyme along with complex effects on the subsequent steps of folding the wild-type and mutant RNA.     

Solution structure and metal-ion binding of the P4 element from bacterial RNase P RNA

We determined the solution structure of two 27-nt RNA hairpins and their complexes with cobalt(III)-hexammine (Co(NH3)3+(6)) by NMR spectroscopy. The RNA hairpins used in this study are the P4 region from Escherichia coli RNase P RNA and a C-to-U mutant that confers altered divalent metal-ion specificity (Ca2+ replaces Mg2+) for catalytic activity of this ribozyme. Co(NH3)3+(6) is a useful spectroscopic probe for Mg(H2O)2+(6)-binding sites because both complexes have octahedral symmetry and have similar radii. The thermodynamics of binding to both RNA hairpins was studied using chemical shift changes upon titration with Mg2+, Ca2+, and Co(NH3)3+(6). We found that the equilibrium binding constants for each of the metal ions was essentially unchanged when the P4 model RNA hairpin was mutated, although the NMR structures show that the RNA hairpins adopt different conformations. In the C-to-U mutant a C.G base pair is replaced by U.G, and the conserved bulged uridine in the P4 wild-type stem shifts in the 3' direction by 1 nt. Intermolecular NOE cross-peaks between Co(NH3)3+(6) and RNA protons were used to locate the site of Co(NH3)3+(6) binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop, but is shifted 5' by more than 1 bp in the mutant. The change of the metal-ion binding site provides a possible explanation for changes in catalytic activity of the mutant RNase P in the presence of Ca2+.     

Structural basis for stabilization of Z-DNA by cobalt hexaammine and magnesium cations

In the equilibrium between B-DNA and Z-DNA in poly(dC-dG), the [Co(NH3)6]3+ ion stabilizes the Z form 4 orders of magnitude more effectively than the Mg2+ ion. The structural basis of this difference is revealed in Z-DNA crystal structures of d(CpGpCpGpCpG) stabilized by either Na+/Mg2+ or Na+/Mg2+ plus [Co(NH3)6]3+. The crystals diffract X-rays to high resolution, and the structures were refined at 1.25 A. The [Co(NH3)6]3+ ion forms five hydrogen bonds onto the surface of Z-DNA, bonding to a guanine O6 and N7 as well as to a phosphate group in the ZII conformation. The Mg2+ ion binds through its hydration shell with up to three hydrogen bonds to guanine N7 and O6. Higher charge, specific fitting of more hydrogen bonds, and a more stable complex all contribute to the great effectiveness of [Co(NH3)6]3+ in stabilizing Z-DNA.     

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.     

EPR spectroscopic analysis of U7 hammerhead ribozyme dynamics during metal ion induced folding

Electron paramagnetic resonance (EPR) spectroscopy was used to examine changes in internal structure and dynamics of the hammerhead ribozyme upon metal ion induced folding, changes in pH, and the presence and absence of ribozyme inhibitors. A nitroxide spin-label was attached to nucleotide U7 of the HH16 catalytic core, and this modified ribozyme was observed to retain catalytic activity. U7 was shown by EPR spectroscopy to be more mobile in the ribozyme-product complex than in either the unfolded ribozyme or the ribozyme-substrate complex. A two-step divalent metal ion dependent folding pathway was observed for the ribozyme-substrate complex with a weak first transition observed at 0.25 mM Mg2+ and a strong second transition observed around 10 mM Mg2+, in agreement with studies using other biophysical and biochemical techniques. Previously, ribozyme activity was observed in the absence of divalent metal ions and the presence of high concentrations of monovalent metal ions, although the activity was less than that observed in the presence of divalent metal ions. Here, we observed similar dynamics for U7 in the presence of 4 M Na+ or Li+, which were distinctively different than that observed in the presence of 10 mM Mg2+, indicating that U7 of the catalytic core forms a different microenvironment under monovalent versus divalent metal ion conditions. Interestingly, the catalytically efficient microenvironment of U7 was similar to that observed in a solution containing 1 M Na+ upon addition of one divalent metal ion per ribozyme. In summary, these results demonstrate that changes in local dynamics, as detected by EPR spectroscopy, can be used to study conformational changes associated with RNA folding and function.     

Essential roles of innersphere metal ions for the formation of the glutamine binding site in a bifunctional ribozyme.

Numerous studies on naturally occurring ribozymes have shown that the functional roles of metal ions in promoting RNA catalysis are diverse. Earlier studies performed on the in vitro selected aminoacyl-transferase ribozyme (ATRib) have revealed that a fully hydrated Mg2+ ion plays an essential role in catalysis [Suga, H., Cowan, J. A., and Szostak, J. W. (1998) Biochemistry 28, 10118-10125]. More recently, we have evolved this ATRib into a bifunctional ribozyme, called AD02 [Lee, N., et al. (2000) Nat. Struct. Biol. 7, 28-33]. This new ribozyme consists of two catalytic domains, the original ATRib domain and a new glutamine-recognition (QR) domain, and exhibits a function of charging glutamine to tRNA. Here we elucidate crucial roles of metal ions involved in the QR domain, that are distinct from those in the ATRib domain. The metal ions in the QR domain require innersphere coordinations, and both Mg2+ and Ca2+ can support catalysis. Extensive Tb3+-Mg2+ and Tb3+-Co(NH3)6(3+) competition cleavage experiments have shown that the QR domain has high and low affinity metal binding sites, which are involved in the Mg2+-dependent structural alteration to form the glutamine binding site [Lee, N., and Suga, H. (2001) RNA 7, 1043-1051]. Kinetic studies in the presence of divalent and monovalent ions have suggested that the essential role of the metal ions in the QR domain is most likely structural.     

Two major tertiary folding transitions of the Tetrahymena catalytic RNA.

The L-21 Tetrahymena ribozyme, an RNA molecule with sequence-specific endoribonuclease activity derived from a self-splicing group I intron, provides a model system for studying the RNA folding problem. A 160 nucleotide, independently folding domain of tertiary structure (the P4-P6 domain) comprises about half of the ribozyme. We now apply Fe(II)-EDTA cleavage to mutants of the ribozyme to explore the role of individual structural elements in tertiary folding of the RNA at equilibrium. Deletion of peripheral elements near the 3' end of the ribozyme destabilizes a region of the catalytic core (P3-P7) without altering the folding of the P4-P6 domain. Three different mutations within the P4-P6 domain that destabilize its folding also shift the folding of the P3-P7 region of the catalytic core to higher MgCl2 concentrations. We conclude that the role of the extended P4-P6 domain and of the 3'-terminal peripheral elements is at least in part to stabilize the catalytic core. The organization of RNA into independently folding domains of tertiary structure may be common in large RNAs, including ribosomal RNAs. Furthermore, the observation of domain-domain interactions in a catalytic RNA supports the feasibility of a primitive spliceosome without any proteins.     

Metal ion requirements for structure and catalysis of an RNA ligase ribozyme

The class I ligase, a ribozyme previously isolated from random sequence, catalyzes a reaction similar to RNA polymerization, positioning its 5'-nucleotide via a Watson-Crick base pair, forming a 3',5'-phosphodiester bond between its 5'-nucleotide and the substrate, and releasing pyrophosphate. Like most ribozymes, it requires metal ions for structure and catalysis. Here, we report the ionic requirements of this self-ligating ribozyme. The ligase requires at least five Mg(2+) for activity and has a [Mg(2+)](1/2) of 70-100 mM. It has an unusual specificity for Mg(2+); there is only marginal activity in Mn(2+) and no detectable activity in Ca(2+), Sr(2+), Ba(2+), Zn(2+), Co(2+), Cd(2+), Pb(2+), Co(NH(3))(6)(3+), or spermine. All tested cations other than Mg(2+), including Mn(2+), inhibit the ribozyme. Hill analysis in the presence of inhibitory cations suggested that Ca(2+) and Co(NH(3))(6)(3+) inhibit by binding at least two sites, but they appear to productively fill a subset of the required sites. Inhibition is not the result of a significant structural change, since the ribozyme assumes a nativelike structure when folded in the presence of Ca(2+) or Co(NH(3))(6)(3+), as observed by hydroxyl-radical mapping. As further support for a nativelike fold in Ca(2+), ribozyme that has been prefolded in Ca(2+) can carry out the self-ligation very quickly upon the addition of Mg(2+). Ligation rates of the prefolded ribozyme were directly measured and proceed at 800 min(-1) at pH 9.0.     

Demonstration of cooperating alpha subunits in working (Na+ + K+)-ATPase by the use of the MgATP complex analogue cobalt tetrammine ATP

The MgATP complex analogue cobalt-tetrammine-ATP [Co(NH3)4ATP] inactivates (Na+ + K+)-ATPase at 37 degrees C slowly in the absence of univalent cations. This inactivation occurs concomitantly with incorporation of radioactivity from [alpha-32P]Co(NH3)4ATP and from [gamma-32P]Co(NH3)4ATP into the alpha subunit. The kinetics of inactivation are consistent with the formation of a dissociable complex of Co(NH3)4ATP with the enzyme (E) followed by the phosphorylation of the enzyme: (Formula: see text). The dissociation constant of the enzyme-MgATP analogue complex at 37 degrees C is Kd = 500 microM, the inactivation rate constant k2 = 0.05 min-1. ATP protects the enzyme against the inactivation by Co(NH3)4ATP due to binding at a site from which it dissociates with a Kd of 360 microM. It is concluded, therefore, that Co(NH3)4ATP binds to the low-affinity ATP binding site of the E2 conformational state. K+, Na+ and Mg2+ protect the enzyme against the inactivation by Co(NH3)4ATP. Whilst Na+ or Mg2+ decrease the inactivation rate constant k2, K+ exerts its protective effect by increasing the dissociation constant of the enzyme.Co(NH3)4ATP complex. The Co(NH3)4ATP-inactivated (Na+ + K+)-ATPase, in contrast to the non-inactivated enzyme, incorporates [3H]ouabain. This indicates that the Co(NH3)4ATP-inactivated enzyme is stabilized in the E2 conformational state. Despite the inactivation of (Na+ + K+)-ATPase by Co(NH3)4ATP from the low-affinity ATP binding site, there is no change in the capacity of the high-affinity ATP binding site (Kd = 0.9 microM) nor of its capability to phosphorylate the enzyme Na+-dependently. Since (Na+ + K+)-ATPase is phosphorylated Na+-dependently from the high-affinity ATP binding site although the catalytic cycle is arrested in the E2 conformational state by specific modification of the low-affinity ATP binding site, it is concluded that both ATP binding sites coexist at the same time in the working sodium pump. This demonstration of interacting catalytic subunits in the E1 and E2 conformational states excludes the proposal that a single catalytic subunit catalyzes (Na+ + K+)-transport.     

A single metal ion plays structural and chemical roles in an aminoacyl-transferase ribozyme.

Catalytically active RNA molecules rely on metal ions for structural and/or catalytic functions. Our in vitro selected aminoacyl-transferase ribozyme is no exception, as it employs a single fully hydrated Mg2+ ion for catalysis [Suga, H., et al. (1998) Biochemistry 37, 10118-10125]. Here we report the essential catalytic residues of the ribozyme and their spatial arrangement in the relation to the metal binding site. Evidence obtained using a combination of Pb2+ and Tb3+ hydrolytic cleavage assays on wild type and mutant ribozymes revealed a cooperative metal binding site that consists of the tandem G:U wobble pairs in P1 and consecutive G:U and U:A pairs in P3. The formation of this concerted Mg2+ binding site positions the P1 and P3 helices in a parallel manner, placing the L3 tetraloop in close proximity to the internal guide sequence (IGS, substrate binding site), which is adjacent to P1. Certain monovalent metal ions inhibit catalysis at low concentrations but support catalysis at high concentrations. These analyses imply that the Mg2+ ion plays both structural and chemical roles and that it brings about the significant rate acceleration in aminoacyl-transfer in concert with the L3-IGS long-range interaction.     

Structure and thermodynamics of metal binding in the P5 helix of a group I intron ribozyme.

The solution structure of an RNA hairpin modelling the P5 helix of a group I intron, complexed with Co(NH3)63+, has been determined by nuclear magnetic resonance. Co(NH3)63+, which possesses a geometry very close to Mg(H2O)62+, was used to identify and characterize a Mg2+binding site in the RNA. Strong and positive intermolecular nuclear Overhauser effect (NOE) cross-peaks define a specific complex in which the Co(NH3)63+molecule is in the major groove of tandem G.U base-pairs. The structure of the RNA is characterized by a very low twist angle between the two G.U base-pairs, providing a flat and narrowed major groove. The Co(NH3)63+, although highly localized, is free to rotate to hydrogen bond in several ways to the O4 atoms of the uracil bases and to N7 and O6 of the guanine bases. Negative and small NOE cross-peaks to other protons in the sequence reveal a non-specific or delocalized interaction, characterized by a high mobility of the cobalt ion. Mn2+titrations of P5 show specific broadening of protons of the G.U base-pairs that form the metal ion binding site, in agreement with the NOE data from Co(NH3)63+. Binding constants for the interaction of Co(NH3)63+and of Mg2+to P5 were determined by monitoring imino proton chemical shifts during titration of the RNA with the metal ions. Dissociation constants are on the order of 0.1 mM for Co(NH3)63+and 1 mM for Mg2+. Binding studies were done on mutants with sequences corresponding to the three orientations of tandem G.U base-pairs. The affinities of Co(NH3)63+and Mg2+for the tandem G.U base-pairs depend strongly on their sequences; the differences can be understood in terms of the different structures of the corresponding metal ion-RNA complexes. Substitution of G.C or A.U for G.U pairs also affected the binding, as expected. These structural and thermodynamic results provide systematic new information about major groove metal ion binding in RNA.     

A Y form of hammerhead ribozyme trapped by photo-cross-links retains full cleavage activity.

The conformation in solution of a small bipartite I-III hammerhead ribozyme has been deduced from the photo-crosslinks formed between cleavable ribo-deoxysubstrates appropriately substituted with the probe deoxy-4-thiouridine and ribozyme residues. The ribozyme-substrate complex is able to adopt a Y-like structure with stems I and II in close proximity in the presence of 400 mM Na+ only. Indeed, a cross-link joining stem I (1.6) to loop II (AL2.4) forms in significant amount under these conditions. This cross-linked complex furthermore elicits, upon Mg2+ addition, a catalytic activity similar to that exhibited by the complexes cross-linked at the distal ends of either stem I or stem III or of the non-substituted bipartite complex. This shows that the reaction mechanism is fully compatible with a strong structural constraint between stems I and II and that sodium ions at high concentration (400 mM) are able to promote a proper folding of hammerhead ribozymes. None of the multiple cross-links formed within the ribozyme core (probe in position 16.1 or 1.1) was found catalytically active. The cross-link patterns nevertheless indicate a higher flexibility of the core in Na+ than in Mg2+. While most of the cross-links can be accommodated by the Y solution structure, some of them (16.1 to U4 and 2.1) definitely can not, suggesting that additional alternative inactive conformations exist in solution.     

The hammerhead cleavage reaction in monovalent cations

Recently, Murray et al. (Chem Biol, 1998, 5:587-595) found that the hammerhead ribozyme does not require divalent metal ions for activity if incubated in high (> or =1 M) concentrations of monovalent ions. We further characterized the hammerhead cleavage reaction in the absence of divalent metal. The hammerhead is active in a wide range of monovalent ions, and the rate enhancement in 4 M Li+ is only 20-fold less than that in 10 mM Mg2+. Among the Group I monovalent metals, rate correlates in a log-linear manner with ionic radius. The pH dependence of the reaction is similar in 10 mM Mg2+, 4 M Li+, and 4 M Na+. The exchange-inert metal complex Co(NH3)3+ also supports substantial hammerhead activity. These results suggest that a metal ion does not act as a base in the reaction, and that the effects of different metal ions on hammerhead cleavage rates primarily reflect structural contributions to catalysis.