Comparison of the hammerhead cleavage reactions stimulated by monovalent and divalent cations

Although the hammerhead reaction proceeds most efficiently in divalent cations, cleavage in 4 M LiCl is only approximately 10-fold slower than under standard conditions of 10 mM MgCl2 (Murray et al., Chem Biol, 1998, 5:587-595; Curtis & Bartel, RNA, 2001, this issue, pp. 546-552). To determine if the catalytic mechanism with high concentrations of monovalent cations is similar to that with divalent cations, we compared the activities of a series of modified hammerhead ribozymes in the two ionic conditions. Nearly all of the modifications have similar deleterious effects under both reaction conditions, suggesting that the hammerhead adopts the same general catalytic structure with both monovalent and divalent cations. However, modification of three ligands previously implicated in the binding of a functional divalent metal ion have substantially smaller effects on the cleavage rate in Li+ than in Mg2+. This result suggests that an interaction analogous to the interaction made by this divalent metal ion is absent in the monovalent reaction. Although the contribution of this divalent metal ion to the overall reaction rate is relatively modest, its presence is needed to achieve the full catalytic rate. The role of this ion appears to be in facilitating formation of the active structure, and any direct chemical role of metal ions in hammerhead catalysis is small.     

The activity and reaction specificity of tyrosine phenol-lyase regulated by monovalent cations

This work was aimed at studying the effect of monovalent inorganic cations (Li+, Na+, K+, Rb+, Cs+, NH+4) on the catalytic and spectral characteristics of tyrosine phenol-lyase from Citrobacter intermedius. These cations were shown to influence the proportion of the beta-elimination reaction rate to the rate of side transamination reaction. Most of the monovalent cations are non-competitive activators of the beta-elimination reaction; Li+ exerts no effect on the enzyme activity in this reaction; Na+ is an inhibitor of the beta-elimination reaction. The activation of tyrosine phenol-lyase by monovalent cations stems from the creation of an active holoenzyme form (lambda max 420 nm) due to conformational rearrangements of the protein molecule.     

Role of divalent metal ions in the hammerhead RNA cleavage reaction.

A hammerhead self-cleaving domain composed of two oligoribonucleotides was used to study the role of divalent metal ions in the cleavage reaction. Cleavage rates were measured as a function of MgCl2, MnCl2, and CaCl2 concentration in the absence or presence of spermine. In the presence of spermine, the rate vs metal ion concentration curves are broader, and lower concentrations of divalent ions are necessary for catalytic activity. This suggests that spermine can promote proper folding of the hammerhead and one or more divalent ions are required for the reaction. Six additional divalent ions were tested for their ability to support hammerhead cleavage. In the absence of spermine, rapid cleavage was observed with Co2+ while very slow cleavage occurred with Sr2+ and Ba2+. No detectable specific cleavage was observed with Cd2+, Zn2+, or Pb2+. However, in the presence of 0.5 mM spermine, rapid cleavage was observed with Zn2+ and Cd2+, and the rate with Sr2+ was increased, indicating that while these three ions could not promote proper folding of the hammerhead they were able to stimulate cleavage. These results suggest certain divalent ions either participate directly in the cleavage mechanism or are specifically involved in stabilizing the tertiary structure of the hammerhead. Additionally, an altered divalent metal ion specificity was observed when a unique phosphorothioate linkage was inserted at the cleavage site. The substitution of a sulfur for a nonbridging oxygen atom substantially reduced the affinity of an important Mg2+ ion necessary for efficient cleavage. In contrast, the reaction proceeds normally with Mn2+, presumably due to its ability to coordinate with both oxygen and sulfur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct reaction pathway promoted by non-divalent-metal cations in a tertiary stabilized hammerhead ribozyme

Divalent ion sensitivity of hammerhead ribozymes is significantly reduced when the RNA structure includes appropriate tertiary stabilization. Therefore, we investigated the activity of the tertiary stabilized "RzB" hammerhead ribozyme in several nondivalent ions. Ribozyme RzB is active in spermidine and Na(+) alone, although the cleavage rates are reduced by more than 1,000-fold relative to the rates observed in Mg(2+) and in transition metal ions. The trivalent cobalt hexammine (CoHex) ion is often used as an exchange-inert analog of hydrated magnesium ion. Trans-cleavage rates exceeded 8 min(-1) in 20 mM CoHex, which promoted cleavage through outersphere interactions. The stimulation of catalysis afforded by the tertiary structural interactions within RzB does not require Mg(2+), unlike other extended hammerhead ribozymes. Site-specific interaction with at least one Mg(2+) ion is suggested by CoHex competition experiments. In the presence of a constant, low concentration of Mg(2+), low concentrations of CoHex decreased the rate by two to three orders of magnitude relative to the rate in Mg(2+) alone. Cleavage rates increased as CoHex concentrations were raised further, but the final fraction cleaved was lower than what was observed in CoHex or Mg(2+) alone. These observations suggest that Mg(2+) and CoHex compete for binding and that they cause misfolded structures when they are together. The results of this study support the existence of an alternate catalytic mechanism used by nondivalent ions (especially CoHex) that is distinct from the one promoted by divalent metal ions, and they imply that divalent metals influence catalysis through a specific nonstructural role.     

Requirement of monovalent cations in the acrosome reaction of guinea pig spermatozoa

The majority of the spermatozoa precapacitated in Ca²⁺-free medium underwent the acrosome raction rapidly when they were transferred to Ca²⁺-containing medium. The presence of Na⁺ and Ca²⁺ in the medium was essential for the acrosome reaction. The vast majority of spermatozoa failed to undergo the reaction in Ca²⁺ medium lacking monovalent ions, although they remained motile. At the concentration of 140 mM, Na⁺, K⁺, Rb⁺, and Cs⁺ all supported the reaction at the maximum level, but at 50 mM the latter three ions were not as effective as Na⁺. Li⁺ was least effective in supporting the reaction. Virtually no acrosome reactions took place when precapacitated spermatozoa were first exposed to Na⁺ medium (no Ca²⁺) and then to Ca²⁺ medium (no Na⁺). On the other hand, a considerably higher proportion of spermatozoa acrosome reacted when they were exposed to these media in the reverse order. The most efficient acrosome reactions took place when the medium contained both a monovalent ion (Na⁺) and Ca²⁺ simultaneously. Possible mechanisms by which monovalent and divalent cations participate in the acrosome reaction are discussed.     

HDV ribozyme activity in monovalent cations

Activity of the two ribozymes from hepatitis delta virus in monovalent salts was examined and compared to activity in Mg2+. Both ribozymes self-cleaved in high concentrations of monovalent cations, and an active site cytosine was required for cleavage activity under those conditions. Cleavage rates were 30-50-fold higher for reactions in LiCl than for reactions in NaCl or NH4Cl, and a thio effect indicated that chemistry was rate-determining for cleavage of the HDV genomic ribozyme in LiCl. Still, in LiCl, there was a more than 100-fold increase in the rate when MgCl2 was included in the reaction. However, the pH-rate profiles for the reactions in LiCl with and without MgCl2 were both bell-shaped with the pH optima in the neutral range. These findings support the idea that monovalent cations can partially substitute for divalent metal ions in the HDV ribozymes, although a divalent metal ion is more effective in supporting catalysis. The absence of a dramatic change in the general shape of pH-rate profiles in LiCl, relative to the profile for reactions including Mg2+, is in contrast to earlier data for the reactions in NaCl and limits our interpretation of the specific role played by the divalent metal ion in the catalytic mechanism.     

Action of metal ions directly involved in the cleavage reaction of hammerhead ribozymes.

Recently, hammerhead ribozyme-mediated cleavage was analyzed as a function of the concentration of La3+ ions in the presence of a fixed concentration of Mg2+ ions so that the role could be monitored of metal ions that are directly involved in the cleavage reaction. The resultant bell-shaped curve for activation of cleavage was used to support the proposed double-metal-ion mechanism of catalysis. However, other studies demonstrated that binding of a metal ion to the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of nucleotide A9 and N7 of nucleotide G10.1 is critical for efficient catalysis. In order to clarify the effect of this metal ion, we chemically synthesized hammerhead ribozyme (7-deaza-R34) that included a minimal modification, namely, an N7-deazaguanine residue in place of G10.1.     

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.     

The effect of monovalent cations on calcium efflux in yeasts

The properties of the calcium efflux system in the yeast Saccharomyces cerevisiae were investigated. After growing the cells overnight in medium containing ⁴⁵Ca, the cells were transferred to medium containing glucose, Hepes buffer (pH 5.2) and monovalent cations. The presence of potassium or sodium in the medium induced efflux of calcium from the cells. The magnitude of the efflux was dependent on the concentration of these cations in the medium. The time course of calcium efflux was analyzed, and two types of exchangeable calcium pools, which turned over at different rates, were detected: ‘Fast turnover’ and ‘slow turnover’. Increase in the concentration of monovalent cations in the medium caused an increase in the fraction of cellular calcium which turned over at a fast rate, and activation of calcium efflux from the ‘slow turnover’ calcium pool. The specific changes in the parameters of calcium efflux induced by monovalent cations were different from those reported previously to be induced by divalent cations. Both processes, i.e. activation of calcium efflux by monovalent and by divalent cations, were found to be additive, indicating that they operate via different mechanisms. Experiments using the respiratory inhibitor Antimycin A, showed that stimulation of calcium efflux by monovalent cations is energy dependent. Lanthanum ions which are known to inhibit calcium influx into yeast cells, inhibitted the activation of calcium efflux by both divalent and monovalent cations. Determination of the cationic composition of the cells indicated that the stimulation of calcium efflux was accompanied by influx of potassium or sodium into the cells.     

Coupling of fast and slow modes in the reaction pathway of the minimal hammerhead ribozyme cleavage

By employing classical molecular dynamics, correlation analysis of coupling between slow and fast dynamical modes, and free energy (umbrella) sampling using classical as well as mixed quantum mechanics molecular mechanics force fields, we uncover a possible pathway for phosphoryl transfer in the self-cleaving reaction of the minimal hammerhead ribozyme. The significance of this pathway is that it initiates from the minimal hammerhead crystal structure and describes the reaction landscape as a conformational rearrangement followed by a covalent transformation. The delineated mechanism is catalyzed by two metal (Mg(2+)) ions, proceeds via an in-line-attack by CYT 17 O2' on the scissile phosphorous (ADE 1.1 P), and is therefore consistent with the experimentally observed inversion configuration. According to the delineated mechanism, the coupling between slow modes involving the hammerhead backbone with fast modes in the cleavage site appears to be crucial for setting up the in-line nucleophilic attack.     

Binary hammerhead ribozymes with high cleavage activity

Binary hammerhead ribozymes consisted of two oligoribonucleotides capable of assembling into hammerhead structure (without loop II) on the RNA target were engineered. Catalytic activities of such ribozymes were investigated in comparison with their full-length analog and ribozyme where two strands were jointed by non-nucleotidic linker. Binary constructs were shown to be significantly more active than the parent full-length hammerhead ribozyme.     

Locating monovalent cations in the grooves of B-DNA

Here we demonstrate that monovalent cations can localize around B-DNA in geometrically regular, sequence-specific sites in oligonucleotide crystals. Positions of monovalent ions were determined from high-resolution X-ray diffraction of DNA crystals grown in the presence of thallium(I) cations (Tl(+)). Tl(+) has previously been shown to be a useful K(+) mimic. Tl(+) positions determined by refinement of model to data are consistent with positions determined using isomorphous F(Tl) - F(K) difference Fouriers and anomalous difference Fouriers. None of the observed Tl(+) sites surrounding CGCGAATTCGCG are fully occupied by Tl(+) ions. The most highly occupied sites, located within the G-tract major groove, have estimated occupancies ranging from 20% to 35%. The occupancies of the minor groove sites are estimated to be around 10%. The Tl(+) positions in general are not in direct proximity to phosphate groups. The A-tract major groove appears devoid of localized cations. The majority of the observed Tl(+) ions interact with a single duplex and so are not engaged in lattice interactions or crystal packing. The locations of the cation sites are dictated by coordination geometry, electronegative potential, avoidance of electropositive amino groups, and cation-pi interactions. It appears that partially dehydrated monovalent cations, hydrated divalent cations, and polyamines compete for a common binding region on the floor of the G-tract major groove.     

Regulation of connexin hemichannels by monovalent cations

Opening of connexin hemichannels in the plasma membrane is highly regulated. Generally, depolarization and reduced extracellular Ca2+ promote hemichannel opening. Here we show that hemichannels formed of Cx50, a principal lens connexin, exhibit a novel form of regulation characterized by extraordinary sensitivity to extracellular monovalent cations. Replacement of extracellular Na+ with K+, while maintaining extracellular Ca2+ constant, resulted in >10-fold potentiation of Cx50 hemichannel currents, which reversed upon returning to Na+. External Cs+, Rb+, NH4+, but not Li+, choline, or TEA, exhibited a similar effect. The magnitude of potentiation of Cx50 hemichannel currents depended on the concentration of extracellular Ca2+, progressively decreasing as external Ca2+ was reduced. The primary effect of K+ appears to be a reduction in the ability of Ca2+, as well as other divalent cations, to close Cx50 hemichannels. Cx46 hemichannels exhibited a modest increase upon substituting Na+ with K+. Analyses of reciprocal chimeric hemichannels that swap NH2- and COOH-terminal halves of Cx46 and Cx50 demonstrate that the difference in regulation by monovalent ions in these connexins resides in the NH2-terminal half. Connexin hemichannels have been implicated in physiological roles, e.g., release of ATP and NAD+ and in pathological roles, e.g., cell death through loss or entry of ions and signaling molecules. Our results demonstrate a new, robust means of regulating hemichannels through a combination of extracellular monovalent and divalent cations, principally Na+, K+, and Ca2+.     

The permeability of endplate channels to monovalent and divalent metal cations

The relative permeability of endplate channels to monovalent and divalent metal ions was determined from reversal potentials. Thallium is the most permeant ion with a permeability ratio relative to Na+ of 2.5. The selectivity among alkali metals is weak with a sequence, Cs+ greater than Rb+ greater than K+ greater than Na+ greater than Li+, and permeability ratios of 1.4, 1.3, 1.1, 1.0, and 0.9. The selectivity among divalent ions is also weak, with a sequence for alkaline earths of Mg++ greater than Ca++ greater than Ba++ greater than Sr++. The transition metal ions Mn++, Co++, Ni++, Zn++, and Cd++ are also permeant. Permeability ratios for divalent ions decreased as the concentration of divalent ion was increased in a manner consistent with the negative surface potential theory of Lewis (1979 J. Physiol. (Lond.). 286: 417--445). With 20 mM XCl2 and 85.5 mM glucosamine.HCl in the external solution, the apparent permeability ratios for the alkaline earth cations (X++) are in the range 0.18--0.25. Alkali metal ions see the endplate channel as a water-filled, neutral pore without high-field-strength sites inside. Their permeability sequence is the same as their aqueous mobility sequence. Divalent ions, however, have a permeability sequence almost opposite from their mobility sequence and must experience some interaction with groups in the channel. In addition, the concentrations of monovalent and divalent ions are increased near the channel mouth by a weak negative surface potential.     

Kinetics of intermolecular cleavage by hammerhead ribozymes.

The hammerhead catalytic RNA effects cleavage of the phosphodiester backbone of RNA through a transesterification mechanism that generates products with 2'-3'-cyclic phosphate and 5'-hydroxyl termini. A minimal kinetic mechanism for the intermolecular hammerhead cleavage reaction includes substrate binding, cleavage, and product release. Elemental rate constants for these steps were measured with six hammerhead sequences. Changes in substrate length and sequence had little effect on the rate of the cleavage step, but dramatic differences were observed in the substrate dissociation and product release steps that require helix-coil transitions. Rates of substrate binding and product dissociation correlated well with predictions based on the behavior of simple RNA duplexes, but substrate dissociation rates were significantly faster than expected. Ribozyme and substrate alterations that eliminated catalytic activity increased the stability of the hammerhead complex. These results suggest that substrate destabilization may play a role in hammerhead catalysis.