Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage.

BACKGROUND: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg(2+)-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1-2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD). The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III. RESULTS: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn(2+). The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage. On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg(2+) ion, which provides the first glimpse of RNase III in action. CONCLUSIONS: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a "ball-and-socket" junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn(2+) binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center.     

Mutational analysis of the nuclease domain of Escherichia coli ribonuclease III. Identification of conserved acidic residues that are important for catalytic function in vitro

The ribonuclease III superfamily represents a structurally distinct group of double-strand-specific endonucleases with essential roles in RNA maturation, RNA decay, and gene silencing. Bacterial RNase III orthologs exhibit the simplest structures, with an N-terminal nuclease domain and a C-terminal double-stranded RNA-binding domain (dsRBD), and are active as homodimers. The nuclease domain contains conserved acidic amino acids, which in Escherichia coli RNase III are E38, E41, D45, E65, E100, D114, and E117. On the basis of a previously reported crystal structure of the nuclease domain of Aquifex aeolicus RNase III, the E41, D114, and E117 side chains of E. coli RNase III are expected to be coordinated to a divalent metal ion (Mg(2+) or Mn(2+)). It is shown here that the RNase III[E41A] and RNase III[D114A] mutants exhibit catalytic activities in vitro in 10 mM Mg(2+) buffer that are comparable to that of the wild-type enzyme. However, at 1 mM Mg(2+), the activities are significantly lower, which suggests a weakened affinity for metal. While RNase III[E41A] and RNase III[D114A] have K(Mg) values that are approximately 2.8-fold larger than the K(Mg) of RNase III (0.46 mM), the RNase III[E41A/D114A] double mutant has a K(Mg) of 39 mM, suggesting a redundant function for the two side chains. RNase III[E38A], RNase III[E65A], and RNase III[E100A] also require higher Mg(2+) concentrations for optimal activity, with RNase III[E100A] exhibiting the largest K(Mg). RNase III[D45A], RNase III[D45E], and RNase III[D45N] exhibit negligible activities, regardless of the Mg(2+) concentration, indicating a stringent functional requirement for an aspartate side chain. RNase III[D45E] activity is partially rescued by Mn(2+). The potential functions of the conserved acidic residues are discussed in the context of the crystallographic data and proposed catalytic mechanisms.     

Intermediate states of ribonuclease III in complex with double-stranded RNA

Bacterial ribonuclease III (RNase III) can affect RNA structure and gene expression in either of two ways: as a processing enzyme that cleaves double-stranded (ds) RNA, or as a binding protein that binds but does not cleave dsRNA. We previously proposed a model of the catalytic complex of RNase III with dsRNA based on three crystal structures, including the endonuclease domain of RNase III with and without bound metal ions and a dsRNA binding protein complexed with dsRNA. We also reported a noncatalytic assembly observed in the crystal structure of an RNase III mutant, which binds but does not cleave dsRNA, complexed with dsRNA. We hypothesize that the RNase III*dsRNA complex can exist in two functional forms, a catalytic complex and a noncatalytic assembly, and that in between the two forms there may be intermediate states. Here, we present four crystal structures of RNase III complexed with dsRNA, representing possible intermediates.     

Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III

Members of the ribonuclease III (RNase III) family are double-stranded RNA (dsRNA) specific endoribonucleases characterized by a signature motif in their active centers and a two-base 3' overhang in their products. While Dicer, which produces small interfering RNAs, is currently the focus of intense interest, the structurally simpler bacterial RNase III serves as a paradigm for the entire family. Here, we present the crystal structure of an RNase III-product complex, the first catalytic complex observed for the family. A 7 residue linker within the protein facilitates induced fit in protein-RNA recognition. A pattern of protein-RNA interactions, defined by four RNA binding motifs in RNase III and three protein-interacting boxes in dsRNA, is responsible for substrate specificity, while conserved amino acid residues and divalent cations are responsible for scissile-bond cleavage. The structure reveals a wealth of information about the mechanism of RNA hydrolysis that can be extrapolated to other RNase III family members.     

Noncatalytic assembly of ribonuclease III with double-stranded RNA

Ribonuclease III (RNase III) represents a family of double-stranded RNA (dsRNA) endonucleases. The simplest bacterial enzyme contains an endonuclease domain (endoND) and a dsRNA binding domain (dsRBD). RNase III can affect RNA structure and gene expression in either of two ways: as a dsRNA-processing enzyme that cleaves dsRNA, or as a dsRNA binding protein that binds but does not cleave dsRNA. We previously determined the endoND structure of Aquifex aeolicus RNase III (Aa-RNase III) and modeled a catalytic complex of full-length Aa-RNase III with dsRNA. Here, we present the crystal structure of Aa-RNase III in complex with dsRNA, revealing a noncatalytic assembly. The major differences between the two functional forms of RNase III.dsRNA are the conformation of the protein and the orientation and location of dsRNA. The flexibility of a 7 residue linker between the endoND and dsRBD enables the transition between these two forms.     

Intrinsic double-stranded-RNA processing activity of Escherichia coli ribonuclease III lacking the dsRNA-binding domain.

The ribonuclease III superfamily represents a structurally related group of double-strand (ds) specific endoribonucleases which play key roles in diverse prokaryotic and eukaryotic RNA maturation and degradation pathways. A dsRNA-binding domain (dsRBD) is a conserved feature of the superfamily and is important for substrate recognition. RNase III family members also exhibit a "catalytic" domain, in part defined by a set of highly conserved amino acids, of which at least one (a glutamic acid) is important for cleavage but not for substrate binding. However, it is not known whether the catalytic domain requires the dsRBD for activity. This report shows that a truncated form of Escherichia coli RNase III lacking the dsRBD (RNase III[DeltadsRBD]) can accurately cleave small processing substrates in vitro. Optimal activity of RNase III[DeltadsRBD] is observed at low salt concentrations (<60 mM Na(+)), either in the presence of Mg(2+) (>25 mM) or Mn(2+) ( approximately 5 mM). At 60 mM Na(+) and 5 mM Mn(2+) the catalytic efficiency of RNase III[DeltadsRBD] is similar to that of RNase III at physiological salt concentrations and Mg(2+). In the presence of Mg(2+) RNase III[DeltadsRBD] is less efficient than the wild-type enzyme, due to a higher K(m). Similar to RNase III, RNase III[DeltadsRBD] is inhibited by high concentrations of Mn(2+), which is due to metal ion occupancy of an inhibitory site on the enzyme. RNase III[DeltadsRBD] retains strict specificity for dsRNA, as indicated by its inability to cleave (rA)(25), (rU)(25), or (rC)(25). Moreover, dsDNA, ssDNA, or an RNA-DNA hybrid are not cleaved. Low (micromolar) concentrations of ethidium bromide block RNase III[DeltadsRBD] cleavage of substrate, which is similar to the inhibition seen with RNase III and is indicative of an intercalative mode of inhibition. Finally, RNase III[DeltadsRBD] is sensitive to specific Watson-Crick base-pair substitutions which also inhibit RNase III. These findings support an RNase III mechanism of action in which the catalytic domain (i) can function independently of the dsRBD, (ii) is dsRNA-specific, and (iii) participates in cleavage site selection.     

Heterodimer-based analysis of subunit and domain contributions to double-stranded RNA processing by Escherichia coli RNase III in vitro

Members of the RNase III family are the primary cellular agents of dsRNA (double-stranded RNA) processing. Bacterial RNases III function as homodimers and contain two dsRBDs (dsRNA-binding domains) and two catalytic sites. The potential for functional cross-talk between the catalytic sites and the requirement for both dsRBDs for processing activity are not known. It is shown that an Escherichia coli RNase III heterodimer that contains a single functional wt (wild-type) catalytic site and an inactive catalytic site (RNase III[E117A/wt]) cleaves a substrate with a single scissile bond with a k(cat) value that is one-half that of wt RNase III, but exhibits an unaltered K(m). Moreover, RNase III[E117A/wt] cleavage of a substrate containing two scissile bonds generates singly cleaved intermediates that are only slowly cleaved at the remaining phosphodiester linkage, and in a manner that is sensitive to excess unlabelled substrate. These results demonstrate the equal probability, during a single binding event, of placement of a scissile bond in a functional or nonfunctional catalytic site of the heterodimer and reveal a requirement for substrate dissociation and rebinding for cleavage of both phosphodiester linkages by the mutant heterodimer. The rate of phosphodiester hydrolysis by RNase III[E117A/wt] has the same dependence on Mg(2+) ion concentration as that of the wt enzyme, and exhibits a Hill coefficient (h) of 2.0+/-0.1, indicating that the metal ion dependence essentially reflects a single catalytic site that employs a two-Mg(2+)-ion mechanism. Whereas an E. coli RNase III mutant that lacks both dsRBDs is inactive, a heterodimer that contains a single dsRBD exhibits significant catalytic activity. These findings support a reaction pathway involving the largely independent action of the dsRBDs and the catalytic sites in substrate recognition and cleavage respectively.     

Stepwise analyses of metal ions in RNase H catalysis from substrate destabilization to product release

In two-metal catalysis, metal ion A has been proposed to activate the nucleophile and metal ion B to stabilize the transition state. We recently reported crystal structures of RNase H-RNA/DNA substrate complexes obtained at 1.5-2.2 Angstroms. We have now determined and report here structures of reaction intermediate and product complexes of RNase H at 1.65-1.85 Angstroms. The movement of the two metal ions suggests how they may facilitate RNA hydrolysis during the catalytic process. Firstly, metal ion A may assist nucleophilic attack by moving towards metal ion B and bringing the nucleophile close to the scissile phosphate. Secondly, metal ion B transforms from an irregular coordination in the substrate complex to a more regular geometry in the product complex. The exquisite sensitivity of Mg(2+) to the coordination environment likely destabilizes the enzyme-substrate complex and reduces the energy barrier to form product. Lastly, product release probably requires dissociation of metal ion A, which is inhibited by either high concentrations of divalent cations or mutation of an assisting protein residue.     

Mechanism of action of Escherichia coli ribonuclease III. Stringent chemical requirement for the glutamic acid 117 side chain and Mn2+ rescue of the Glu117Asp mutant

Escherichia coli ribonuclease III (EC 3.1.24) is a double-strand- (ds-) specific endoribonuclease involved in the maturation and decay of cellular, phage, and plasmid RNAs. RNase III is a homodimer and requires Mg(2+) to hydrolyze phosphodiesters. The RNase III polypeptide contains an N-terminal catalytic (nuclease) domain which exhibits eight highly conserved acidic residues, at least one of which (Glu117) is important for phosphodiester hydrolysis but not for substrate binding [Li and Nicholson (1996) EMBO J. 15, 1421-1433]. To determine the side chain requirements for activity, Glu117 was changed to glutamine or aspartic acid. The mutant proteins were purified as (His)(6)-tagged species, and both exhibited normal homodimeric behavior as shown by chemical cross-linking. The Glu117Gln mutant is unable to cleave substrate in vitro under all tested conditions but can bind substrate. The Glu117Asp mutant also is defective in cleavage while able to bind substrate. However, low level activity is observed at extended reaction times and high enzyme concentrations, with an estimated catalytic efficiency approximately 15 000-fold lower than that of RNase III. The activity of the Glu117Asp mutant but not that of the Glu117Gln mutant can be greatly enhanced by substituting Mn(2+) for Mg(2+), with the catalytic efficiency of the Glu117Asp-Mn(2+) holoenzyme approximately 400-fold lower than that of the RNase III-Mn(2+) holoenzyme. For RNase III, a Mn(2+) concentration of 1 mM provides optimal activity, while concentrations >5 mM are inhibitory. In contrast, the Glu117Asp mutant is not inhibited by high concentrations of Mn(2+). Finally, high concentrations of Mg(2+) do not inhibit RNase III nor relieve the Mn(2+)-dependent inhibition. In summary, these experiments establish the stringent functional requirement for a precisely positioned carboxylic acid group at position 117 and reveal two classes of divalent metal ion binding sites on RNase III. One site binds either Mg(2+) or Mn(2+) and supports catalysis, while the other site is specific for Mn(2+) and confers inhibition. Glu117 is important for the function of both sites. The implications of these findings on the RNase III catalytic mechanism are discussed.     

Structure of the active subunit of the yeast exosome core, Rrp44: diverse modes of substrate recruitment in the RNase II nuclease family

The eukaryotic exosome is a macromolecular complex essential for RNA processing and decay. It has recently been shown that the RNase activity of the yeast exosome core can be mapped to a single subunit, Rrp44, which processively degrades single-stranded RNAs as well as RNAs containing secondary structures. Here we present the 2.3 A resolution crystal structure of S. cerevisiae Rrp44 in complex with single-stranded RNA. Although Rrp44 has a linear domain organization similar to bacterial RNase II, in three dimensions the domains have a different arrangement. The three domains of the classical nucleic-acid-binding OB fold are positioned on the catalytic domain such that the RNA-binding path observed in RNase II is occluded. Instead, RNA is threaded to the catalytic site via an alternative route suggesting a mechanism for RNA-duplex unwinding. The structure provides a molecular rationale for the observed biochemical properties of the RNase R family of nucleases.     

Solution structure of RNase P RNA

The ribonucleoprotein enzyme ribonuclease P (RNase P) processes tRNAs by cleavage of precursor-tRNAs. RNase P is a ribozyme: The RNA component catalyzes tRNA maturation in vitro without proteins. Remarkable features of RNase P include multiple turnovers in vivo and ability to process diverse substrates. Structures of the bacterial RNase P, including full-length RNAs and a ternary complex with substrate, have been determined by X-ray crystallography. However, crystal structures of free RNA are significantly different from the ternary complex, and the solution structure of the RNA is unknown. Here, we report solution structures of three phylogenetically distinct bacterial RNase P RNAs from Escherichia coli, Agrobacterium tumefaciens, and Bacillus stearothermophilus, determined using small angle X-ray scattering (SAXS) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis. A combination of homology modeling, normal mode analysis, and molecular dynamics was used to refine the structural models against the empirical data of these RNAs in solution under the high ionic strength required for catalytic activity.     

Insights into RNA/DNA hybrid recognition and processing by RNase H from the crystal structure of a non-specific enzyme-dsDNA complex

Ribonuclease HI (RNase H) is a member of the nucleotidyl-transferase superfamily and endo-nucleolytically cleaves the RNA portion in RNA/DNA hybrids and removes RNA primers from Okazaki fragments. The enzyme also binds RNA and DNA duplexes but is unable to cleave either. Three-dimensional structures of bacterial and human RNase H catalytic domains bound to RNA/DNA hybrids have revealed the basis for substrate recognition and the mechanism of cleavage. In order to visualize the enzyme’s interactions with duplex DNA and to establish the structural differences that afford tighter binding to RNA/DNA hybrids relative to dsDNA, we have determined the crystal structure of Bacillus halodurans RNase H in complex with the B-form DNA duplex [d(CGCGAATTCGCG)]2. The structure demonstrates that the inability of the enzyme to cleave DNA is due to the deviating curvature of the DNA strand relative to the substrate RNA strand and the absence of Mg2+ at the active site. A subset of amino acids engaged in contacts to RNA 2′-hydroxyl groups in the substrate complex instead bind to bridging or non-bridging phosphodiester oxygens in the complex with dsDNA. Qualitative comparison of the enzyme’s interactions with the substrate and inhibitor duplexes is consistent with the reduced binding affinity for the latter and sheds light on determinants of RNase H binding and cleavage specificity.     

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.     

Cooperative RNP assembly: complementary rescue of structural defects by protein and RNA subunits of archaeal RNase P

Ribonuclease P (RNase P) is a ribonucleoprotein complex that utilizes a Mg(2+)-dependent RNA catalyst to cleave the 5' leader of precursor tRNAs (pre-tRNAs) and generate mature tRNAs. The bacterial RNase P protein (RPP) aids RNase P RNA (RPR) catalysis by promoting substrate binding, Mg(2+) coordination and product release. Archaeal RNase P comprises an RPR and at least four RPPs, which have eukaryal homologs and function as two binary complexes (POP5·RPP30 and RPP21·RPP29). Here, we employed a previously characterized substrate-enzyme conjugate [pre-tRNA(Tyr)-Methanocaldococcus jannaschii (Mja) RPR] to investigate the functional role of a universally conserved uridine in a bulge-helix structure in archaeal RPRs. Deletion of this bulged uridine resulted in an 80-fold decrease in the self-cleavage rate of pre-tRNA(Tyr)-MjaΔU RPR compared to the wild type, and this defect was partially ameliorated upon addition of either RPP pair. The catalytic defect in the archaeal mutant RPR mirrors that reported in a bacterial RPR and highlights a parallel in their active sites. Furthermore, an N-terminal deletion mutant of Pyrococcus furiosus (Pfu) RPP29 that is defective in assembling with its binary partner RPP21, as assessed by isothermal titration calorimetry and NMR spectroscopy, is functional when reconstituted with the cognate Pfu RPR. Collectively, these results indicate that archaeal RPPs are able to compensate for structural defects in their cognate RPR and vice-versa, and provide striking examples of the cooperative subunit interactions critical for driving archaeal RNase P toward its functional conformation.     

Evidence for induced fit in bacterial RNase P RNA-mediated cleavage

RNase P with its catalytic RNA subunit is involved in the processing of a number of RNA precursors with different structures. However, precursor tRNAs are the most abundant substrates for RNase P. Available data suggest that a tRNA is folded into its characteristic structure already at the precursor state and that RNase P recognizes this structure. The tRNA D-/T-loop domain (TSL-region) is suggested to interact with the specificity domain of RNase P RNA while residues in the catalytic domain interact with the cleavage site. Here, we have studied the consequences of a productive interaction between the TSL-region and its binding site (TBS) in the specificity domain using tRNA precursors and various hairpin-loop model substrates. The different substrates were analyzed with respect to cleavage site recognition, ground-state binding, cleavage as a function of the concentration of Mg(2+) and the rate of cleavage under conditions where chemistry is suggested to be rate limiting using wild-type Escherichia coli RNase P RNA, M1 RNA, and M1 RNA variants with structural changes in the TBS-region. On the basis of our data, we conclude that a productive TSL/TBS interaction results in a conformational change in the M1 RNA substrate complex that has an effect on catalysis. Moreover, it is likely that this conformational change comprises positioning of chemical groups (and Mg(2+)) at and in the vicinity of the cleavage site. Hence, our findings are consistent with an induced-fit mechanism in RNase P RNA-mediated cleavage.     

Terbium-mediated footprinting probes a catalytic conformational switch in the antigenomic hepatitis delta virus ribozyme

The two forms of the hepatitis delta virus ribozyme are derived from the genomic and antigenomic RNA strands of the human hepatitis delta virus (HDV), where they serve a crucial role in pathogen replication by catalyzing site-specific self-cleavage reactions. The HDV ribozyme requires divalent metal ions for formation of its tertiary structure, consisting of a tight double-nested pseudoknot, and for efficient self- (or cis-) cleavage. Comparison of recently solved crystal structures of the cleavage precursor and 3' product indicates that a significant conformational switch is required for catalysis by the genomic HDV ribozyme. Here, we have used the lanthanide metal ion terbium(III) to footprint the precursor and product solution structures of the cis-acting antigenomic HDV ribozyme. Inhibitory Tb(3+) binds with high affinity to similar sites on RNA as Mg(2+) and subsequently promotes slow backbone scission. We find subtle, yet significant differences in the terbium(III) footprinting pattern between the precursor and product forms of the antigenomic HDV ribozyme, consistent with differences in conformation as observed in the crystal structures of the genomic ribozyme. In addition, UV melting profiles provide evidence for a less tight tertiary structure in the precursor. In both the precursor and product we observe high-affinity terbium(III) binding sites in joining sequence J4/2 (Tb(1/2) approximately 4 microM) and loop L3, which are key structural components forming the catalytic core of the HDV ribozyme, as well as in several single-stranded regions such as J1/2 and the L4 tetraloop (Tb(1/2) approximately 50 microM). Sensitized luminescence spectroscopy confirms that there are at least two affinity classes of Tb(3+) binding sites. Our results thus demonstrate that a significant conformational change accompanies catalysis in the antigenomic HDV ribozyme in solution, similar to the catalytic conformational switch observed in crystals of the genomic form, and that structural and perhaps catalytic metal ions bind close to the catalytic core.     

Structural characterization of a ribonuclease III processing signal.

The structure of a ribonuclease III processing signal from bacteriophage T7 was examined by NMR spectroscopy, optical melting, and chemical and enzymatic modification. A 41 nucleotide variant of the T7 R1.1 processing signal has two Watson-Crick base-paired helices separated by an internal loop, consistent with its predicted secondary structure. The internal loop is neither rigidly structured nor completely exposed to solvent, and seems to be helical. The secondary structure of R1.1 RNA is largely insensitive to the monovalent cation concentration, which suggests that the monovalent cation sensitivity of secondary site cleavage by RNase III is not due to a low salt-induced RNA conformational change. However, spectroscopic data show that Mg2+ affects the conformation of the internal loop, suggesting a divalent cation binding site(s) within this region. The Mg(2+)-dependence of RNase III processing of some substrates may reflect not only a requirement for a divalent cation as a catalytic cofactor, but also a requirement for a local RNA conformation which is divalent cation-stabilized.     

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+.