Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modification-interference approach to identify nonbridging phosphate oxygens within 16S rRNA that are important for the in vitro assembly of the Escherichia coli 30S small ribosomal subunit and for its association with the 50S large ribosomal subunit. The 30S small subunit was reconstituted from phosphorothioate-substituted 16S rRNA and small subunit proteins. Active 30S subunits were selected by their ability to bind to the 50S large subunit and form 70S ribosomes. Analysis of the selected population shows that phosphate oxygens at specific positions in the 16S rRNA are important for either subunit assembly or for binding to the 50S subunit. The X-ray crystallographic structures of the 30S subunit suggest that some of these phosphate oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature 30S subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any interaction in the mature 30S subunit, suggesting that they play a role in the early steps of the 30S subunit assembly.     

Solution conformation of various uridine diphosphoglucose salts as probed by NMR spectroscopy

The solution conformations of uridine diphosphoglucose (UDP-Glc) under a variety of conditions (solvent, ionic strength, various mono- and divalent cations) have been studied by NMR spectroscopy (1H, 13C, 31P, and 25Mg). In the case of divalent cations (Ca2+, Mg2+, Mn2+) the phosphate oxygens are the preferred coordination sites and analysis of the 25Mg linewidths of solutions with various [Mg2+]/[UDP-Glc] ratios, indicates that the 1:1 Mg2+ UDP-Glc complex is the major species. From 13C relaxation data and hydrodynamic theory, it has been demonstrated that under all conditions UDP-Glc adopts a fairly extended overall shape and that magnesium ions lead to a significant increase in the average length of the UDP-Glc molecule as compared to monovalent cations. Thus, one of the roles of the metal ion in enzymic reactions involving nucleotide sugars may be to preorganize the nucleotide sugar.     

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.     

Specific phosphorothioate substitutions probe the active site of Bacillus subtilis ribonuclease P

Ribonuclease P (RNase P) is a ribonucleoprotein that requires magnesium ions to catalyze the 5' maturation of transfer RNA. To identify interactions essential for catalysis, the properties of RNase P containing single sulfur substitutions for nonbridging phosphodiester oxygens in helix P4 of Bacillus subtilis RNase P were analyzed using transient kinetic experiments. Sulfur substitution at the nonbridging oxygens of the phosphodiester bond of nucleotide U51 only modestly affects catalysis. However, phosphorothioate substitutions at A49 and G50 decrease the cleavage rate constant enormously (300-4,000-fold for P RNA and 500-15,000-fold for RNase P holoenzyme) in magnesium without affecting the affinity of pre-tRNA(Asp), highlighting the importance of this region for catalysis. Furthermore, addition of manganese enhances pre-tRNA cleavage catalyzed by B. subtilis RNase P RNA containing an Sp phosphorothioate modification at A49, as observed for Escherichia coli P RNA [Christian et al., RNA, 2000, 6:511-519], suggesting that an essential metal ion may be coordinated at this site. In contrast, no manganese rescue is observed for the A49 Sp phosphorothioate modification in RNase P holoenzyme. These differential manganese rescue effects, along with affinity cleavage, suggest that the protein component may interact with a metal ion bound near A49 in helix P4 of P RNA.     

Structural roles of monovalent cations in the HDV ribozyme

The hepatitis delta virus (HDV) ribozyme catalyzes viral RNA self-cleavage through general acid-base chemistry in which an active-site cytidine and at least one metal ion are involved. Monovalent metal ions support slow catalysis and were proposed to substitute for structural, but not catalytic, divalent metal ions in the RNA. To investigate the role of monovalent cations in ribozyme structure and function, we determined the crystal structure of the precursor HDV ribozyme in the presence of thallium ions (Tl(+)). Two Tl(+) ions can occupy a previously observed divalent metal ion hexahydrate-binding site located near the scissile phosphate, but are easily competed away by cobalt hexammine, a magnesium hexahydrate mimic and potent reaction inhibitor. Intriguingly, a third Tl(+) ion forms direct inner-sphere contacts with the ribose 2'-OH nucleophile and the pro-S(p) scissile phosphate oxygen. We discuss possible structural and catalytic implications of monovalent cation binding for the HDV ribozyme mechanism.     

Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations

Accurate predictions of DNA stability in physiological and enzyme buffers are important for the design of many biological and biochemical assays. We therefore investigated the effects of magnesium, potassium, sodium, Tris ions, and deoxynucleoside triphosphates on melting profiles of duplex DNA oligomers and collected large melting data sets. An empirical correction function was developed that predicts melting temperatures, transition enthalpies, entropies, and free energies in buffers containing magnesium and monovalent cations. The new correction function significantly improves the accuracy of predictions and accounts for ion concentration, G-C base pair content, and length of the oligonucleotides. The competitive effects of potassium and magnesium ions were characterized. If the concentration ratio of [Mg (2+)] (0.5)/[Mon (+)] is less than 0.22 M (-1/2), monovalent ions (K (+), Na (+)) are dominant. Effects of magnesium ions dominate and determine duplex stability at higher ratios. Typical reaction conditions for PCR and DNA sequencing (1.5-5 mM magnesium and 20-100 mM monovalent cations) fall within this range. Conditions were identified where monovalent and divalent cations compete and their stability effects are more complex. When duplexes denature, some of the Mg (2+) ions associated with the DNA are released. The number of released magnesium ions per phosphate charge is sequence dependent and decreases surprisingly with increasing oligonucleotide length.     

Control of mitochondrial Mg++-efflux]

Inorganic phosphate stimulates the release of Mg++ from liver mitochondria, depending on concentration; a concentration as low as 0.1 mM phosphate is already effective. The process is dependent on the electron transfer of the respiratory chain, and its rate is highest under conditions of endogenous respiration and with ascorbate and TMPD as substrates, respectively. The phosphate stimulated release of Mg++ is followed, with a pronounced delay, by a Ca++ efflux and a swelling of mitochondria. Addition of EGTA strongly reduced the rate of Mg++ liberation in the presence and absence of inorganic phosphate. Exogenous Ca++ is able to abolish the EGTA effect. ADP and ATP inhibit the phosphate stimulated release of Mg++. Phosphoenol pyruvate and free fatty acids enhance the rate of Mg++ and Ca++ efflux from the mitochondria. The results permit the conclusion that inorganic phosphate, Ca++ and various metabolites of the cell metabolism influence the Mg++ distribution between the extra- and intramitochondrial space, thus controlling the permeability of the mitochondrial inner membrane for monovalent cations.     

Solution probing of metal ion binding by helix 27 from Escherichia coli 16S rRNA

Helix (H)27 from Escherichia coli 16S ribosomal (r)RNA is centrally located within the small (30S) ribosomal subunit, immediately adjacent to the decoding center. Bacterial 30S subunit crystal structures depicting Mg(2+) binding sites resolve two magnesium ions within the vicinity of H27: one in the major groove of the G886-U911 wobble pair, and one within the GCAA tetraloop. Binding of such metal cations is generally thought to be crucial for RNA folding and function. To ask how metal ion-RNA interactions in crystals compare with those in solution, we have characterized, using solution NMR spectroscopy, Tb(3+) footprinting and time-resolved fluorescence resonance energy transfer (tr-FRET), location, and modes of metal ion binding in an isolated H27. NMR and Tb(3+) footprinting data indicate that solution secondary structure and Mg(2+) binding are generally consistent with the ribosomal crystal structures. However, our analyses also suggest that H27 is dynamic in solution and that metal ions localize within the narrow major groove formed by the juxtaposition of the loop E motif with the tandem G894-U905 and G895-U904 wobble pairs. In addition, tr-FRET studies provide evidence that Mg(2+) uptake by the H27 construct results in a global lengthening of the helix. We propose that only a subset of H27-metal ion interactions has been captured in the crystal structures of the 30S ribosomal subunit, and that small-scale structural dynamics afforded by solution conditions may contribute to these differences. Our studies thus highlight an example for differences between RNA-metal ion interactions observed in solution and in crystals.     

Metal ion probing of rRNAs: evidence for evolutionarily conserved divalent cation binding pockets.

Ribosomes are multifunctional RNP complexes whose catalytic activities absolutely depend on divalent metal ions. It is assumed that structurally and functionally important metal ions are coordinated to highly ordered RNA structures that form metal ion binding pockets. One potent tool to identify the structural surroundings of high-affinity metal ion binding pockets is metal ion-induced cleavage of RNA. Exposure of ribosomes to divalent metal ions, such as Pb2+, Mg2+, Mn2+, and Ca2+, resulted in site-specific cleavage of rRNAs. Sites of strand scission catalyzed by different cations accumulate at distinct positions, indicating the existence of general metal ion binding centers in the highly folded rRNAs in close proximity to the cleavage sites. Two of the most efficient cleavage sites are located in the 5' domain of both 23S and 16S rRNA, regions that are known to self-fold even in the absence of ribosomal proteins. Some of the efficient cleavage sites were mapped to the peptidyl transferase center located in the large ribosomal subunit. Furthermore, one of these cleavages was clearly diminished upon AcPhe-tRNA binding to the P site, but was not affected by uncharged tRNA. This provides evidence for a close physical proximity of a metal ion to the amino acid moiety of charged tRNAs. Interestingly, comparison of the metal ion cleavage pattern of eubacterial 70S with that of human 80S ribosomes showed that certain cleavage sites are evolutionarily highly conserved, thus demonstrating an identical location of a nearby metal ion. This suggests that cations, bound to evolutionarily constrained binding sites, are reasonable candidates for being of structural or functional importance.     

Solution structure and thermodynamics of a divalent metal ion binding site in an RNA pseudoknot.

Identification and characterization of a metal ion binding site in an RNA pseudoknot was accomplished using cobalt (III) hexammine, Co(NH3)63+, as a probe for magnesium (II) hexahydrate, Mg(H2O)62+, in nuclear magnetic resonance (NMR) structural studies. The pseudoknot causes efficient -1 ribosomal frameshifting in mouse mammary tumor virus. Divalent metal ions, such as Mg2+, are critical for RNA structure and function; Mg2+preferentially stabilizes the pseudoknot relative to its constituent hairpins. The use of Co(NH3)63+as a substitute for Mg2+was investigated by ultraviolet absorbance melting curves, NMR titrations of the imino protons, and analysis of NMR spectra in the presence of Mg2+or Co (NH3)63+. The structure of the pseudoknot-Co(NH3)63+complex reveals an ion-binding pocket formed by a short, two-nucleotide loop and the major groove of a stem. Co(NH3)63+stabilizes the sharp loop-to-stem turn and reduces the electrostatic repulsion of the phosphates in three proximal strands. Hydrogen bonds are identified between the Co(NH3)63+protons and non-bridging phosphate oxygen atoms, 2' hydroxyl groups, and nitrogen and oxygen acceptors on the bases. The binding site is significantly different from that previously characterized in the major groove surface of tandem G.U base-pairs, but is similar to those observed in crystal structures of a fragment of the 5 S rRNA and the P5c helix of the Tetrahymena thermophila group I intron. Changes in chemical shifts occurred at the same pseudoknot protons on addition of Mg2+as on addition of Co(NH3)63+, indicating that both ions bind at the same site. Ion binding dissociation constants of approximately 0.6 mM and 5 mM (in 200 mM Na+and a temperature of 15 degrees C) were obtained for Co(NH3)63+and Mg2+, respectively, from the change in chemical shift as a function of metal ion concentration. An extensive array of non-sequence-specific hydrogen bond acceptors coupled with conserved structural elements within the binding pocket suggest a general mode of divalent metal ion stabilization of this type of frameshifter pseudoknot. These results provide new thermodynamic and structural insights into the role divalent metal ions play in stabilizing RNA tertiary structural motifs such as pseudoknots.     

Stabilization of RNA tertiary structure by monovalent cations

The effects of monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), and NH4(+)) on the thermal stability of RNA tertiary structure were investigated by UV melting. We show that with the RNA used here (nucleotides 1051-1108 of Escherichia coli 23 S rRNA with four base substitutions), monovalent cations and Mg(2+) compete in stabilizing the RNA tertiary structure, and that the competition takes place between two boundaries: one where Mg(2+) concentration is zero and the other where it is maximally stabilizing ("saturating"). The pattern of competition is the same for all monovalent cations and depends on the cation's ability to displace Mg(2+) from the RNA, its ability to stabilize tertiary structure in the absence of Mg(2+), and its ability to stabilize tertiary structure at saturating Mg(2+) concentrations. The stabilizing ability of a monovalent cation depends on its unhydrated ionic radius, and at a low monovalent cation concentration and saturating Mg(2+), there is a (calculated) net release of a single monovalent cation/RNA molecule when tertiary structure is denatured. The implications are that under these conditions there is at least one binding site for monovalent cations on the RNA, the site is specifically associated with formation of stable tertiary structure, K(+) is the most effective of the tested cations, and Mg(2+) appears ineffective at this site. At high ionic strength, and in the absence of Mg(2+), stabilization of tertiary structure is still monovalent-cation specific and ionic-radius dependent, but a larger number of cations ( approximately eight) are released upon RNA tertiary structure denaturation, and NH(4)(+) appears to be the most effective cation in stabilizing tertiary structure under these conditions. In the majority of the experiments, methanol was added as a cosolvent to the buffer. Its use allowed the examination of the behavior of monovalent ions under conditions where their effects would otherwise have been too weak to be observed. Methanol stabilizes tertiary but not secondary structure of the RNA. There was no evidence that it either causes qualitative changes in cation-binding properties of the RNA or a change in the pattern of monovalent cation/Mg(2+) competition.     

Magnesium ion induced proton release as a probe for the polyelectrolytic structure of ribosomal RNAs and subunits

E coli ribosomes and rRNA's released 20 to 50 protons upon jump of magnesium ion concentration from 1 mM to 20 mM. The Mg2+-induced proton release was measured separately for 16S rRNA, 23S rRNA, 30S subunit, and 50S subunit by a new spectrophotometric method that had a much better sensitivity than the pH-stat method. The proton release from the subunits and rRNA's were similar in the number of protons, the pH dependence that had a minimum at neutral pH, and the upward concaveness of the Scatchard plot. From these results, the main source of protons in ribosomal subunits was assigned to nucleotide bases of rRNA's that showed a downward pKa shift upon Mg2+-ion binding. The subunits and rRNA's, however, differed in the proton release. 16S rRNA released protons somewhat more effectively than 23S rRNA, while 30S subunit released protons 2 to 5 times more effectively than 50S subunit. The marked difference between the two subunits suggest that ionizable bases in 16S and 23S rRNA's are covered and their pKa values are shifted by ribosomal proteins to different extents. The association of 30S and 50S subunits induced little proton release, showing that few ionizable groups with pKa near neutral pH are involved in the association. E. coli tRNA and poly U also showed Mg2+-induced proton release. The amounts of protons released from rRNA's, tRNA, and poly U were roughly proportional to the amount of bases not hydrogen bonded. The Mg2+-induced proton release from the natural and synthetic RNA's can be explained by the electrostatic field effect of polyphosphate backbones on bases not hydrogen bonded, as proposed in a previous paper. It also reflects the conformational structure of each RNA molecule.     

Three metal ions participate in the reaction catalyzed by T5 flap endonuclease

Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a "two-metal-ion mechanism." Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of > 4 A appears incompatible with this mechanism. To clarify the roles played by FEN metal ions, we report here a detailed evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg2+ or Mn2+). The apparent substrate association constant is maximized by binding two magnesium ions. This may reflect a metal-dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN primarily uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions.     

The 1.3 A resolution structure of the RNA tridecamer r(GCGUUUGAAACGC): metal ion binding correlates with base unstacking and groove contraction

Metal ions play a key role in RNA folding and activity. Elucidating the rules that govern the binding of metal ions is therefore an essential step for better understanding the RNA functions. High-resolution data are a prerequisite for a detailed structural analysis of ion binding on RNA and, in particular, the observation of monovalent cations. Here, the high-resolution crystal structures of the tridecamer duplex r(GCGUUUGAAACGC) crystallized under different conditions provides new structural insights on ion binding on GAAA/UUU sequences that exhibit both unusual structural and functional properties in RNA. The present study extends the repertory of RNA ion binding sites in showing that the two first bases of UUU triplets constitute a specific site for sodium ions. A striking asymmetric pattern of metal ion binding in the two equivalent halves of the palindromic sequence demonstrates that sequence and its environment act together to bind metal ions. A highly ionophilic half that binds six metal ions allows, for the first time, the observation of a disodium cluster in RNA. The comparison of the equivalent halves of the duplex provides experimental evidences that ion binding correlates with structural alterations and groove contraction.     

Magnesium binding to yeast ribosomes

This paper describes a theoretical and experimental analysis of the binding of magnesium ions to yeast, ribosomes. In the theoretical considerations the interactions between charges located on a macroion are included. In the calculations these interactions result in a term, in which both the charge and the radius of the macroion are accounted for. It appears that on dissociation of the ribosomes both the charge and the radius change, but in such a way, that the term, which accounts for the electrostatic interactions, remains constant. As a consequence the dissociation can lie neglected in the analyses of the binding experiments. Our experiments indicate that two binding reactions between ribosomes and magnesium ions occur. The endpoints of these reactions correspond to about 0.40 and 1.0 equivalent magnesium per ribosomal phosphate, respectively. The pK values are about 3.8 and 2.2, respectively. The experimental results indicate that the effect, of monovalent cations can be explained as a pure ionic strength effect, though the binding of monovalent cations could not be excluded completely.     

Binding of magnesium ions and ethidium bromide: comparison of ribosomes and free ribosomal RNA.

Comparative studies of free ribosomal RNA and ribosomes were made with two probes, Mg++ ions and ethidium bromide, which interact with RNA in different ways. Mg++. E. coli 16 S rRNA and 30 S ribosomes were equilibrated with four different buffers. Equilibration required several days at 4 degrees and several hours at 37 degrees. In all buffers ribosomes bound more Mg than free rRNA, the difference sometimes reaching 20--30%. Ribosomes were more resistant than free rRNA to heat denaturation and their denaturation was more highly cooperative. Ribosomes that bound more Mg++ had higher denaturation temperatures. Ethidium bromide. Fluorescence enhancement studies of ethidium intercalation showed the free 16 S rRNA to have 50--80 binding sites per molecule. A large fraction of these sites were present and accessible in the ribosome, but their ethidium-binding constants were reduced by an order of magnitude. In addition, free rRNA contained a small number of very strong binding sites that were virtually absent in the ribosomes.     

An energy dependent divalent cation-hydrogen ion binding in the outer membranes of rat liver mitochondria and its dependence on monovalent cation-hydrogen ion binding

Intact mitochondria are able to bind monovalent and divalent metal cations and to release protons in an energy-independent exchange process. Directly accessible binding sites exist in the outer membrane. They seem to be identical for monovalent and divalent metal ions. The inner membrane-matrix-fraction possesses exchange sites after ultrasonic disruption only for monovalent cations, but not for divalent cations.     

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.     

Ion Condensation onto Ribozyme Is Site Specific and Fold Dependent

The highly charged RNA molecules, with each phosphate carrying a single negative charge, cannot fold into well-defined architectures with tertiary interactions in the absence of ions. For ribozymes, divalent cations are known to be more efficient than monovalent ions in driving them to a compact state, although Mg²⁺ ions are needed for catalytic activities. Therefore, how ions interact with RNA is relevant in understanding RNA folding. It is often thought that most of the ions are territorially and nonspecifically bound to the RNA, as predicted by the counterion condensation theory. Here, we show using simulations of Azoarcus ribozyme, based on an accurate coarse-grained three-site interaction model with explicit divalent and monovalent cations, that ion condensation is highly specific and depends on the nucleotide position. The regions with high coordination between the phosphate groups and the divalent cations are discernible even at very low Mg²⁺ concentrations when the ribozyme does not form tertiary interactions. Surprisingly, these regions also contain the secondary structural elements that nucleate subsequently in the self-assembly of RNA, implying that ion condensation is determined by the architecture of the folded state. These results are in sharp contrast to interactions of ions (monovalent and divalent) with rigid charged rods, in which ion condensation is uniform and position independent. The differences are explained in terms of the dramatic nonmonotonic shape fluctuations in the ribozyme as it folds with increasing Mg²⁺ or Ca²⁺ concentration.     

A single nucleotide linked to a switch in metal ion reactivity preference in the HDV ribozymes

The two ribozymes of hepatitis delta virus (HDV) cleave faster in divalent metal ions than in monovalent cations, and a variety of divalent metal ions can act as catalysts in supporting these higher rates. Although the ribozymes are closely related in sequence and structure, they display a different metal ion preference; the genomic form cleaves moderately faster in Mg2+ than in Ca2+ while the reverse is true for the antigenomic ribozyme. This difference raises questions about understanding the catalytic role of the metal ion in the reaction. We found that the metal ion reactivity preference correlated with the identity of a single nucleotide 5' of the cleavage site (-1 position). It is a U in the genomic sequence and a C in the antigenomic sequence. With both ribozymes, the reactivity preference for Mg2+ and Ca2+ could be reversed with a change at this position (C to U or U to C). Moreover, with an A at position -1, there was a relative increase in cleavage rates in low concentrations of Mn2+ for both ribozymes. Metal ion reactivity preference was also linked to changes in pH, and the pH-rate profiles could be shifted with nucleotide changes at position -1. Together, the data provide biochemical evidence in support of an organized active site, as seen in the crystal structures, where at least one metal ion, an ionizable group, and the conformation of the phosphate backbone at the cleavage site interact in concert to promote cleavage.