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.     

Evidence that binding of C5 protein to P RNA enhances ribozyme catalysis by influencing active site metal ion affinity

The RNA subunit (P RNA) of the bacterial RNase P ribonucleoprotein is a ribozyme that catalyzes the Mg-dependent hydrolysis of pre-tRNA, but it requires an essential protein cofactor (P protein) in vivo that enhances substrate binding affinities and catalytic rates in a substrate dependent manner. Previous studies of Bacillus subtilis RNase P, containing a Type B RNA subunit, showed that its cognate protein subunit increases the affinity of metal ions important for catalysis, but the functional role of these ions is unknown. Here, we demonstrate that the Mg2+ dependence of the catalytic step for Escherichia coli RNase P, which contains a more common Type A RNA subunit, is also modulated by its cognate protein subunit (C5), indicating that this property is fundamental to P protein. To monitor specifically the binding of active site metal ions, we analyzed quantitatively the rescue by Cd2+ of an inhibitory Rp phosphorothioate modification at the pre-tRNA cleavage site. The results show that binding of C5 protein increases the apparent affinity of the rescuing Cd2+, providing evidence that C5 protein enhances metal ion affinity in the active site, and thus is likely to contribute significantly to rate enhancement at physiological metal ion concentrations.     

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+.     

Cation-specific structural accommodation within a catalytic RNA

Metal ions facilitate the folding of the hairpin ribozyme but do not participate directly in catalysis. The metal complex cobalt(III) hexaammine supports folding and activity of the ribozyme and also mediates specific internucleotide photocrosslinks, several of which retain catalytic ability. These crosslinks imply that the active core structure organized by [Co(NH3)6]3+ is different from that organized by Mg2+ and that revealed in the crystal structure [Rupert, P. B., and Ferre-D'Amare, A. R. (2001) Nature 410, 780-786] (1). Residues U+2 and C+3 of the substrate, in particular, adopt different conformations in [Co(NH3)6]3+. U+2 is bulged out of loop A and stacked on residue G36, whereas the nucleotide at position +3 is stacked on G8, a nucleobase crucial for catalysis. Cleavage kinetics performed with +2 variants and a C+3 U variant correlate with the crosslinking observations. Variants that decreased cleavage rates in magnesium up to 70-fold showed only subtle decreases or even increases in observed rates when assayed in [Co(NH3)6]3+. Here, we propose a model of the [Co(NH3)6]3+-mediated catalytic core generated by MC-SYM that is consistent with these data.     

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.     

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.     

Ribonuclease III cleavage of a bacteriophage T7 processing signal. Divalent cation specificity, and specific anion effects.

Escherichia coli ribonuclease III, purified to homogeneity from an overexpressing bacterial strain, exhibits a high catalytic efficiency and thermostable processing activity in vitro. The RNase III-catalyzed cleavage of a 47 nucleotide substrate (R1.1 RNA), based on the bacteriophage T7 R1.1 processing signal, follows substrate saturation kinetics, with a Km of 0.26 microM, and kcat of 7.7 min.-1 (37 degrees C, in buffer containing 250 mM potassium glutamate and 10 mM MgCl2). Mn2+ and Co2+ can support the enzymatic cleavage of the R1.1 RNA canonical site, and both metal ions exhibit concentration dependences similar to that of Mg2+. Mn2+ and Co2+ in addition promote enzymatic cleavage of a secondary site in R1.1 RNA, which is proposed to result from the altered hydrolytic activity of the metalloenzyme (RNase III 'star' activity), exhibiting a broadened cleavage specificity. Neither Ca2+ nor Zn2+ support RNase III processing, and Zn2+ moreover inhibits the Mg(2+)-dependent enzymatic reaction without blocking substrate binding. RNase III does not require monovalent salt for processing activity; however, the in vitro reactivity pattern is influenced by the monovalent salt concentration, as well as type of anion. First, R1.1 RNA secondary site cleavage increases as the salt concentration is lowered, perhaps reflecting enhanced enzyme binding to substrate. Second, the substitution of glutamate anion for chloride anion extends the salt concentration range within which efficient processing occurs. Third, fluoride anion inhibits RNase III-catalyzed cleavage, by a mechanism which does not involve inhibition of substrate binding.     

A 2'-methyl or 2'-methylene group at G+1 in precursor tRNA interferes with Mg2+ binding at the enzyme-substrate interface in E-S complexes of E. coli RNase P

We analyzed processing of precursor tRNAs carrying a single 2'-deoxy, 2'-OCH(3), or locked nucleic acid (LNA) modification at G+1 by Escherichia coli RNase P RNA in the absence and presence of its protein cofactor. The extra methyl or methylene group caused a substrate binding defect, which was rescued at higher divalent metal ion (M(2+)) concentrations (more efficiently with Mn(2+) than Mg(2+)), and had a minor effect on cleavage chemistry at saturating M(2+) concentrations. The 2'-OCH(3) and LNA modification at G+1 resulted in higher metal ion cooperativity for substrate binding to RNase P RNA without affecting cleavage site selection. This indicates disruption of an M(2+) binding site in enzyme-substrate complexes, which is compensated for by occupation of alternative M(2+) binding sites of lower affinity. The 2'-deoxy modification at G+1 caused at most a two-fold decrease in the cleavage rate; this mild defect relative to 2'-OCH(3) and LNA at G+1 indicates that the defect caused by the latter two is steric in nature. We propose that the 2'-hydroxyl at G+1 in the substrate is in the immediate vicinity of the M(2+) cluster at the phosphates of A67 to U69 in helix P4 of E. coli RNase P 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.     

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.     

Engineered allosteric ribozymes that respond to specific divalent metal ions

In vitro selection was used to isolate five classes of allosteric hammerhead ribozymes that are triggered by binding to certain divalent metal ion effectors. Each of these ribozyme classes are similarly activated by Mn2+, Fe2+, Co2+, Ni2+, Zn2+ and Cd2+, but their allosteric binding sites reject other divalent metals such as Mg2+, Ca2+ and Sr2+. Through a more comprehensive survey of cations, it was determined that some metal ions (Be2+, Fe3+, Al3+, Ru2+ and Dy2+) are extraordinarily disruptive to the RNA structure and function. Two classes of RNAs examined in greater detail make use of conserved nucleotides within the large internal bulges to form critical structures for allosteric function. One of these classes exhibits a metal-dependent increase in rate constant that indicates a requirement for the binding of two cation effectors. Additional findings suggest that, although complex allosteric functions can be exhibited by small RNAs, larger RNA molecules will probably be required to form binding pockets that are uniquely selective for individual cation effectors.     

Comparative analyses of hairpin substrate recognition by Escherichia coli and Bacillus subtilis ribonuclease P ribozymes

Previously, we reported that the substrate shape recognition of the Escherichia coli ribonuclease (RNase) P ribozyme depends on the concentration of magnesium ion in vitro. We additionally examined the Bacillus subtilis RNase P ribozyme and found that the B. subtilis enzyme also required high magnesium ion, above 10 mM, for cleavage of a hairpin substrate. The results of kinetic studies showed that the metal ion concentration affected both the catalysis and the affinity of the ribozymes toward a hairpin RNA substrate.     

Purification and characterization of RNase P from Clostridium sporogenes

RNase P is a multi-subunit enzyme responsible for the accurate processing of the 5' terminus of all tRNAs. The RNA subunit from Clostridium sporogenes has been partially purified and characterized. The RNA is approximately 400 nucleotides long and makes a precise endonucleolytic cleavage at the mature 5' terminus of tRNA. The RNA requires moderate concentrations of Mg2+ (20 mM) and relatively high concentrations of NH4Cl (800 mM) for optimal activity. Mn2+ effectively substitutes for Mg2+ at 2 mM. Zn2+, Ni2+, Ca2+, and Co2+ are ineffective at stimulating activity. Monovalent ions are, in general, more effective the greater the ionic radius (NH+4 greater than Cs greater than Rb greater than K greater than Na). In contrast to the activity of Bacillus subtilis, C. sporogenes RNase P RNA is significant more active in (NH4)2SO4 than in NH4Cl.     

Characterization of structure and metal ions specificity of Co2+-binding RNA aptamers

Studies on RNA motifs capable of binding metal ions have largely focused on Mg(2+)-specific motifs, therefore information concerning interactions of other metal ions with RNA is still very limited. Application of the in vitro selection approach allowed us to isolate two RNA aptamers that bind Co(2+) ions. Structural analysis of their secondary structures revealed the presence of two motifs, loop E and "kissing" loop complex, commonly occurring in RNA molecules. The Co(2+)-induced cleavage method was used for identification of Co(2+)-binding sites after the determination of the optimal cleavage conditions. In the aptamers, Co(2+) ions seem to bind to N7 atoms of purines, inducing cleavage of the adjacent phosphodiester bonds, similarly as is the case with yeast tRNA(Phe). Although the in vitro selection experiment was carried out in the presence of Co(2+) ions only, the aptamers displayed broader metal ions specificity. This was shown by inhibition of Co(2+)-induced cleavages in the presence of the following transition metal ions: Zn(2+), Cd(2+), Ni(2+), and Co(NH(3))(6)(3+) complex. On the other hand, alkaline metal ions such as Mg(2+), Ca(2+), Sr(2+), and Ba(2+) affected Co(2+)-induced cleavages only slightly. Multiple metal ions specificity of Co(2+)-binding sites has also been reported for other in vitro selected or natural RNAs. Among many factors that influence metal specificity of the Co(2+)-binding pocket, chemical properties of metal ions, such as their hardness as well as the structure of the coordination site, seem to be particularly important.     

Catalysis by RNase P RNA: unique features and unprecedented active site plasticity

Metal ions are essential cofactors for precursor tRNA (ptRNA) processing by bacterial RNase P. The ribose 2'-OH at nucleotide (nt) -1 of ptRNAs is known to contribute to positioning of catalytic Me2+. To investigate the catalytic process, we used ptRNAs with single 2'-deoxy (2'-H), 2'-amino (2'-N), or 2'-fluoro (2'-F) modifications at the cleavage site (nt -1). 2' modifications had small (2.4-7.7-fold) effects on ptRNA binding to E. coli RNase P RNA in the ground state, decreasing substrate affinity in the order 2'-OH > 2'-F > 2'-N > 2'-H. Effects on the rate of the chemical step (about 10-fold for 2'-F, almost 150-fold for 2'-H and 2'-N) were much stronger, and, except for the 2'-N modification, resembled strikingly those observed in the Tetrahymena ribozyme-catalyzed reaction at corresponding position. Mn2+ rescued cleavage of the 2'-N but also the 2'-H-modified ptRNA, arguing against a direct metal ion coordination at this location. Miscleavage between nt -1 and -2 was observed for the 2'-N-ptRNA at low pH (further influenced by the base identities at nt -1 and +73), suggesting repulsion of a catalytic metal ion due to protonation of the amino group. Effects caused by the 2'-N modification at nt -1 of the substrate allowed us to substantiate a mechanistic difference in phosphodiester hydrolysis catalyzed by Escherichia coli RNase P RNA and the Tetrahymena ribozyme: a metal ion binds next to the 2' substituent at nt -1 in the reaction catalyzed by RNase P RNA, but not at the corresponding location in the Tetrahymena ribozyme reaction.     

Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor. The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper. Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addition of Fe2+ ions to the reaction mixture and was eliminated by treatment of the enzymes with EDTA. The chelated enzymes were reactivated by a variety of divalent metal ions, including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+. The specific activities of the reactivated enzymes varied widely with the different metals as follows: Mn2+ greater than Cd2+, Fe2+ greater than Co2+ greater than Ni2+, Cu2+, Zn2+ much greater than Ca2+. Steady state kinetic analysis of the Mn2+, Fe2+, Co2+, and Zn2+ forms of the phenylalanine-sensitive isozyme (DAHPS(Phe)) revealed that metal variation significantly affected the apparent affinity for the substrate, erythrose 4-phosphate, but not for the second substrate, phosphoenolpyruvate, or for the feedback inhibitor, L-phenylalanine. The tetrameric DAHPS(Phe) exhibited positive homotropic cooperativity with respect to erythrose 4-phosphate, phophoenolpyruvate, and phenylalanine in the presence of all metals tested.     

The naturally trans-acting ribozyme RNase P RNA has leadzyme properties

Divalent metal ions promote hydrolysis of RNA backbones generating 5′OH and 2′;3′P as cleavage products. In these reactions, the neighboring 2′OH act as the nucleophile. RNA catalyzed reactions also require divalent metal ions and a number of different metal ions function in RNA mediated cleavage of RNA. In one case, the LZV leadzyme, it was shown that this catalytic RNA requires lead for catalysis. So far, none of the naturally isolated ribozymes have been demonstrated to use lead to activate the nucleophile. Here we provide evidence that RNase P RNA, a naturally trans-acting ribozyme, has leadzyme properties. But, in contrast to LZV RNA, RNase P RNA mediated cleavage promoted by Pb²⁺ results in 5′ phosphate and 3′OH as cleavage products. Based on our findings, we infer that Pb²⁺ activates H₂O to act as the nucleophile and we identified residues both in the substrate and RNase P RNA that most likely influenced the positioning of Pb²⁺ at the cleavage site. Our data suggest that Pb²⁺ can promote cleavage of RNA by activating either an inner sphere H₂O or a neighboring 2′OH to act as nucleophile.     

The role of divalent cations in structure and function of murine adenosine deaminase.

For murine adenosine deaminase, we have determined that a single zinc or cobalt cofactor bound in a high affinity site is required for catalytic function while metal ions bound at an additional site(s) inhibit the enzyme. A catalytically inactive apoenzyme of murine adenosine deaminase was produced by dialysis in the presence of specific zinc chelators in an acidic buffer. This represents the first production of the apoenzyme and demonstrates a rigorous method for removing the occult cofactor. Restoration to the holoenzyme is achieved with stoichiometric amounts of either Zn2+ or Co2+ yielding at least 95% of initial activity. Far UV CD and fluorescence spectra are the same for both the apo- and holoenzyme, providing evidence that removal of the cofactor does not alter secondary or tertiary structure. The substrate binding site remains functional as determined by similar quenching measured by tryptophan fluorescence of apo- or holoenzyme upon mixing with the transition state analog, deoxycoformycin. Excess levels of adenosine or N6- methyladenosine incubated with the apoenzyme prior to the addition of metal prevent restoration, suggesting that the cofactor adds through the substrate binding cleft. The cations Ca2+, Cd2+, Cr2+, Cu+, Cu2+, Mn2+, Fe2+, Fe3+, Pb2+, or Mg2+ did not restore adenosine deaminase activity to the apoenzyme. Mn2+, Cu2+, and Zn2+ were found to be competitive inhibitors of the holoenzyme with respect to substrate and Cd2+ and Co2+ were noncompetitive inhibitors. Weak inhibition (Ki > or = 1000 microM) was noted for Ca2+, Fe2+, and Fe3+.     

Purine N7 groups that are crucial to the interaction of Escherichia coli rnase P RNA with tRNA.

We have detected by nucleotide analog interference mapping (NAIM) purine N7 functional groups in Escherichia coli RNase P RNA that are important for tRNA binding under moderate salt conditions (0.1 M Mg2+, 0.1 M NH4+). The majority of identified positions represent highly or universally conserved nucleotides. Our assay system allowed us, for the first time, to identify c7-deaza interference effects at two G residues (G292, G306). Several c7-deazaadenine interference effects (A62, A65, A136, A249, A334, A351) have also been identified in other studies performed at very different salt concentrations, either selecting for substrate binding in the presence of 0.025 M Ca2+ and 1 M NH4+ or self-cleavage of a ptRNA-RNase P RNA conjugate in the presence of 3 M NH4+ or Na+. This indicates that these N7 functional groups play a key role in the structural organization of ribozyme-substrate and -product complexes. We further observed that a c7-deaza modification at A76 of tRNA interferes with tRNA binding to and ptRNA processing by E. coli RNase P RNA. This finding combined with the strong c7-deaza interference at G292 of RNase P RNA supports a model in which substrate and product binding to E. coli RNase P RNA involves the formation of intermolecular base triples (A258-G292-C75 and G291-G259-A76).