Thiophilic Metal Ions Research Articles

Catalytic Metal Ion-Substrate Coordination during Nonenzymatic RNA Primer Extension.

Nonenzymatic template-directed RNA copying requires catalysis by divalent metal ions. The primer extension reaction involves the attack of the primer 3'-hydroxyl on the adjacent phosphate of a 5'-5'-imidazolium-bridged dinucleotide substrate. However, the nature of the interaction of the catalytic metal ion with the reaction center remains unclear. To explore the coordination of the catalytic metal ion with the imidazolium-bridged dinucleotide substrate, we examined catalysis by oxophilic and thiophilic metal ions with both diastereomers of phosphorothioate-modified substrates. We show that Mg2+ and Cd2+ exhibit opposite preferences for the two phosphorothioate substrate diastereomers, indicating a stereospecific interaction of the divalent cation with one of the nonbridging phosphorus substituents. High-resolution X-ray crystal structures of the products of primer extension with phosphorothioate substrates reveal the absolute stereochemistry of this interaction and indicate that catalysis by Mg2+ involves inner-sphere coordination with the nonbridging phosphate oxygen in the pro-SP position, while thiophilic cadmium ions interact with sulfur in the same position, as in one of the two phosphorothioate substrates. These results collectively suggest that during nonenzymatic RNA primer extension with a 5'-5'-imidazolium-bridged dinucleotide substrate the interaction of the catalytic Mg2+ ion with the pro-SP oxygen of the reactive phosphate plays a crucial role in the metal-catalyzed SN2(P) reaction.

Prebiotic Environments with Mg2+ and Thiophilic Metal Ions Increase the Thermal Stability of Cysteine and Non-cysteine Peptides.

Wet-dry cycles driven by heating to high temperatures are frequently invoked for the prebiotic synthesis of peptides. Similarly, iron-sulfur clusters are often cited as an example of an ancient catalyst that helped prune early chemical systems into metabolic-like pathways. Because extant iron-sulfur clusters are metallocofactors of protein enzymes and nearly ubiquitous across biology, a reasonable hypothesis is that prebiotic iron-sulfur peptides formed on the early Earth. However, iron-sulfur clusters are coordinated by multiple cysteine residues, and the stability of cysteines to the heat steps of wet-dry cycles has not been determined. It, therefore, has remained unclear if the peptides needed to stabilize the formation of iron-sulfur clusters could have formed. If not, then iron-sulfur-dependent activity may have emerged later, when milder, more biological-like peptide synthesis machinery took hold. Here, we report the thermal stability of cysteine-containing peptides. We show that temperatures of 150 °C lead to the rapid degradation of cysteinyl peptides. However, the presence of Mg2+ at environmentally reasonable concentrations leads to significant protection. Thiophilic metal ions also protect against degradation at 150 °C but require concentrations not frequently observed in the environment. Nevertheless, cysteine-containing peptides are stable at lower, prebiotically plausible temperatures in seawater, carbonate lake, and ferrous lake conditions. The data are consistent with the persistence of cysteine-containing peptides on the early Earth in environments rich in metal ions. High concentrations of Mg2+ are common intra- and extra-cellularly, suggesting that the protection afforded by Mg2+ may reflect conditions that were present on the prebiotic Earth.

Anatase Nanocrystals Covalently Functionalized with EDTA-diol: Interaction with Aromatic Sulfur.

The current study addresses the generation of surface-modified nanograined anatase (5 nm) by a single-step in situ process. The EDTA-diol (ester) functionality existing on the surface of anatase nanoparticles was proved with the help of a battery of physicochemical techniques, including FTIR, XPS, and NMR spectral measurements. The sample showed excellent dispersity in ethanol. The anatase lattice showed disorder and had oxygen vacancies. From the XPS analysis, the existence of 14% of Ti3+ was established. The functionalized sample remained thermally stable up to 250 °C, beyond which it transformed into a graphite-titania nanocomposite. The interfacial ligand (ester) to metal (Ti) charge transfer (LMCT) transitions were present in the UV-visible spectrum of the sample and indicated the functionalization to be covalent. Unsaturated OH-groups on the surface yielded a ζ-potential value of -39.8 mV. The covalently functionalized nanotitania was exploited in the adsorptive desulfurization of thiophene, an aromatic sulfur model compound. The consequences of surface adsorption of thiophene were analyzed with the help of UV-visible, FTIR, and 1H and 13C NMR spectra and EDS and XPS measurements. The adsorption data, derived from the batch process, were fitted successfully to pseudo-second-order kinetics and the Temkin model. The adsorption capacity of covalently functionalized titania was compared with several other high surface area adsorbents containing thiophilic metal ions.

Highly sensitive and selective strategy for imaging Hg2+ using near-infrared squaraine dye in live cells and zebrafish

This article describes the rational design and synthesis of a monothiosquaraine (MTSQ) fluorescent Hg2+ probe that exhibits near-infrared fluorescence, ultrahigh brightness, fast response, high sensitivity and excellent selectivity. Utilizing the strategy of the incorporation of the heavy atom effect and self-assembly caused quenching, the background fluorescence of MTSQ was greatly lowered and the sensitivity in PBS buffer solution was highly enhanced with a very low limit of detection (LOD = 1.2 nM). Systematic studies revealed excellent selectivity and specificity for Hg2+ over other thiophilic metal ions such as Ag+, Pb2+, and Cd2+. The mechanistic investigation indicates that self-assembly and desulfurization of MTSQ are responsible for Hg2+-triggered significantly higher fluorescence enhancement. With high sensitivity, optimal pH range of 3–7, weak background, good water-solubility and low cytotoxicity, MTSQ therefore can be applied for imaging Hg2+ in live cells and zebrafish. The very simple but effective strategy reported here can provide a more convenient approach for designing NIR probes.

Further insights into the metal ion binding abilities and the metalation pathway of a plant metallothionein from Musa acuminata.

The superfamily of metallothioneins (MTs) combines a diverse group of metalloproteins, sharing the characteristics of rather low molecular weight and high cysteine content. The latter provides MTs with the capability to coordinate thiophilic metal ions, in particular those with a d 10 electron configuration. The sub-family of plant MT3 proteins is only poorly characterized and there is a complete lack of three-dimensional structure information. Building upon our previous results on the Musa acuminata MT3 (musMT3) protein, the focus of the present work is to understand the metal cluster formation process, the role of the single histidine residue present in musMT3, and the metal ion binding affinity. We concentrate our efforts on the coordination of ZnII and CdII ions, using CoII as a spectroscopic probe for ZnII binding. The overall protein-fold is analysed with a combination of limited proteolytic digestion, mass spectrometry, and dynamic light scattering. Histidine coordination of metal ions is probed with extended X-ray absorption fine structure spectroscopy and CoII titration experiments. Initial experiments with isothermal titration calorimetry provide insights into the thermodynamics of metal ion binding.

Sunlight and ultrasound-assisted synthesis of photoluminescent silver nanoclusters: A unique ‘Knock out’ sensor for thiophilic metal ions

We demonstrate the fine tuning of sunlight and ultrasound in green synthesis of photoluminescent silver nanoclusters (AgNCs) having quantum dot (QD) character for the first time. The use of hazardous additives (e.g. reducing agents, organic solvents, etc.) is minimized. The role of sunlight (which ruptures the S–S linkage) and ultrasound (which produces free radicals) in controlling various synthetic parameter as well as properties are carefully monitored and scientifically explained. The synthesized materials have the capabilities in sensing thiophilic metals ions (Hg2+, Pb2+, Cd2+, Zn2+) up to nanomolar (nM) and milimolar (mM) ranges by photoluminescence (PL) spectroscopy and PL microscopy respectively. The sensing of metal ions is based on fluorescence quenching. Hard soft acid base principle (HSAB) plays a major role to recognise the synthesized material as a unique ‘knock out’ sensor. The HR-MS and HR-TEM show that the silver nanocluster is made of 2–6 silver atoms having diameter in the range of 2–3nm respectively. The synthesized materials may be a good candidate for bio-labelling, bio-sensing and cell imaging.

Unique fluorogenic ratiometric fluorescent chemodosimeter for rapid sensing of CN(-) in water.

A new benzimidazole-spiropyran conjugate chemosensor molecule (BISP) has been synthesized and characterized by (1)H NMR spectroscopy, mass spectrometry (ESI-MS), and elemental analysis. The two isomeric forms (BISP↔BIMC) were shown to be highly selective and sensitive to CN(-) among the ten anions studied in aqueous HEPES buffer, as shown by fluorescence and absorption spectroscopy and even by visual color changes, with a detection limit of 1.7 μM for BIMC. The reaction of CN(-) with BIMC was monitored by (1)H NMR spectroscopy, high-resolution mass spectrometry (HRMS), UV/Vis measurements, and fluorescence spectroscopy in HEPES buffer of pH 7.4. TDDFT calculations were performed in order to correlate the electronic properties of the chemosensor with its cyanide complex. Further, titration against thiophilic metal ions like Au(3+), Cu(2+), Ag(+), and Hg(2+) with [BIMC-CN] in situ showed that it acts as a secondary recognition ensemble toward Au(3+) and Cu(2+) by switch-on fluorescence. In addition, a reversible logic-gate property of BIMC has been demonstrated through a feedback loop in the presence of CN(-) and Au(3+) ions, respectively. Furthermore, the use of BIMC to detect CN(-) in live cells by fluorescence imaging has also been demonstrated. Notably, test strips based on BIMC were fabricated, which could serve as convenient and efficient CN(-) test kits.

Engineering a gold-specific regulon for cell-based visual detection and recovery of gold

A gold-specific sensory protein GolS from Samonella gol regulon was incorporated into E. coli, which in conjunction with an engineered downstream red fluorescence protein allowed the highly sensitive and selective whole-cell detection of gold(III) ions by naked eyes. The putative gold-chaperone, GolB, in the gol regulon was next verified to be specific to gold(I) ions over other metal ions including copper(I). The subsequent display of GolB on E. coli cell surface permitted selective enrichment of gold ions from media containing various thiophilic metal ions. The cell surface-enriched gold(I) was further shown to be easily recovered and the gold-deprived bacteria were capable for re-usage. E. coli bacteria harboring these gold-specific elements from the gol regulon could be a valuable tool for visual detection and facile recycling of gold ions from environmental resources.

Cofactor-mediated conformational control in the bifunctional kinase/RNase Ire1

BackgroundIre1 is a signal transduction protein in the endoplasmic reticulum (ER) membrane that serves to adjust the protein-folding capacity of the ER according to the needs of the cell. Ire1 signals, in a transcriptional program, the unfolded protein response (UPR) via the coordinated action of its protein kinase and RNase domains. In this study, we investigated how the binding of cofactors to the kinase domain of Ire1 modulates its RNase activity.ResultsOur results suggest that the kinase domain of Ire1 initially binds cofactors without activation of the RNase domain. RNase is activated upon a subsequent conformational rearrangement of Ire1 governed by the chemical properties of bound cofactors. The conformational step can be selectively inhibited by chemical perturbations of cofactors. Substitution of a single oxygen atom in the terminal β-phosphate group of a potent cofactor ADP by sulfur results in ADPβS, a cofactor that binds to Ire1 as well as to ADP but does not activate RNase. RNase activity can be rescued by thiophilic metal ions such as Mn2+ and Cd2+, revealing a functional metal ion-phosphate interaction which controls the conformation and RNase activity of the Ire1 ADP complex. Mutagenesis of the kinase domain suggests that this rearrangement involves movement of the αC-helix, which is generally conserved among protein kinases. Using X-ray crystallography, we show that oligomerization of Ire1 is sufficient for placing the αC-helix in the active, cofactor-bound-like conformation, even in the absence of cofactors.ConclusionsOur structural and biochemical evidence converges on a model that the cofactor-induced conformational change in Ire1 is coupled to oligomerization of the receptor, which, in turn, activates RNase. The data reveal that cofactor-Ire1 interactions occur in two independent steps: binding of a cofactor to Ire1 and subsequent rearrangement of Ire1 resulting in its self-association. The pronounced allosteric effect of cofactors on protein-protein interactions involving Ire1's kinase domain suggests that protein kinases and pseudokinases encoded in metazoan genomes may use ATP pocket-binding ligands similarly to exert signaling roles other than phosphoryl transfer.

Use of divalent metal ions in the DNA cleavage reaction of topoisomerase IV

It has long been known that type II topoisomerases require divalent metal ions in order to cleave DNA. Kinetic, mutagenesis and structural studies indicate that the eukaryotic enzymes utilize a novel variant of the canonical two-metal-ion mechanism to promote DNA scission. However, the role of metal ions in the cleavage reaction mediated by bacterial type II enzymes has been controversial. Therefore, to resolve this critical issue, this study characterized the DNA cleavage reaction of Escherichia coli topoisomerase IV. We utilized a series of divalent metal ions with varying thiophilicities in conjunction with oligonucleotides that replaced bridging and non-bridging oxygen atoms at (and near) the scissile bond with sulfur atoms. DNA scission was enhanced when thiophilic metal ions were used with substrates that contained bridging sulfur atoms. In addition, the metal-ion dependence of DNA cleavage was sigmoidal in nature, and rates and levels of DNA cleavage increased when metal ion mixtures were used in reactions. Based on these findings, we propose that topoisomerase IV cleaves DNA using a two-metal-ion mechanism in which one of the metal ions makes a critical interaction with the 3′-bridging atom of the scissile phosphate and facilitates DNA scission by the bacterial type II enzyme.

Near‐Infrared Cell‐Permeable Hg2+‐Selective Ratiometric Fluorescent Chemodosimeters and Fast Indicator Paper for MeHg+ Based on Tricarbocyanines

Three tricarbocyanine dyes (IR-897, IR-877, and IR-925) with different thiourea substituents that function as dosimeter units through specific Hg(2+)-induced desulfurization have been demonstrated in a fast indicator paper for Hg(2+) and MeHg(+) ions. In comparison with available Hg(2+)-selective chemodosimeters, IR-897 and IR-877 show several advantages, such as convenient synthesis, very long wavelengths falling in the near-infrared (NIR) region (650-900 nm) with high molar extinction coefficients, a ratiometric response, and quite low disturbance with Ag(+) and Cu(2+) ions. They exhibit large redshifts, which result in a clear color change from deep blue to pea green that can be easily monitored by the naked eye for a convenient indicator paper. In emission spectra, they display a characteristic turn-off mode at 780 nm and turn-on mode at 830 nm with titration of Hg(2+) ions. Remarkably, the signal/noise (S/N) ratio with other thiophilic metal ions (Ag(+) and Cu(2+)) is greatly enhanced with ratiometric measurement of two channels: excitation spectra mode (I(810 nm)/I(670 nm), monitored at 830 nm) and emission spectra mode (I(830 nm)/I(780 nm), isosbestic absorption point at 730 nm as excitation). The distinct response is dependent upon the electron-donating effect of the thiourea substituents; that is, the stronger the electron-donating capability of the thiourea substituents, the faster the Hg(2+)-promoted cyclization. Additionally, experiments with living SW1116 cells show that these three tricarbocyanine dyes with low toxicity can exhibit special characteristics that are favorable for visualizing intracellular Hg(2+) and MeHg(+) ions in biological systems, including excellent membrane permeability, minimal interfering absorption and fluorescence from biological samples, low scattering, and deep penetration into tissues.

Human topoisomerase II uses a two-metal-ion mechanism for DNA cleavage

The DNA cleavage reaction of human topoisomerase IIα is critical to all of the physiological and pharmacological functions of the protein. While it has long been known that the type II enzyme requires a divalent metal ion in order to cleave DNA, the role of the cation in this process is not known. To resolve this fundamental issue, the present study utilized a series of divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that replaced the 3′-bridging oxygen of the scissile bond with a sulfur atom (i.e. 3′-bridging phosphorothiolates). Rates and levels of DNA scission were greatly enhanced when thiophilic metal ions were included in reactions that utilized sulfur-containing substrates. Based on these results and those of reactions that employed divalent cation mixtures, we propose that topoisomerase IIα mediates DNA cleavage via a two-metal-ion mechanism. In this model, one of the metal ions makes a critical interaction with the 3′-bridging atom of the scissile phosphate. This interaction greatly accelerates rates of enzyme-mediated DNA cleavage, and most likely is needed to stabilize the leaving 3′-oxygen.

Discrimination in Metal‐Ion Binding to RNA Dinucleotides with a Non‐Bridging Oxygen or Sulfur in the Phosphate Diester Link

Replacement of a non-bridging oxygen in the phosphate diester bond by a sulfur has become quite popular in nucleic acid research and is often used as a probe, for example, in ribozymes, where the normally essential Mg(2+) is partly replaced by a thiophilic metal ion to reactivate the system. Despite these widely applied rescue experiments no detailed studies exist quantifying the affinity of metal ions to such terminal sulfur atoms. Therefore, we performed potentiometric pH titrations to determine the binding properties of pUp((S))U(3-) towards Mg(2+), Mn(2+), Zn(2+), Cd(2+), and Pb(2+), and compared these data with those previously obtained for the corresponding pUpU(3-) complexes. The primary binding site in both dinucleotides is the terminal phosphate group. Theoretically, also the formation of 10-membered chelates involving the terminal oxygen or sulfur atoms of the (thio)phosphate bridge is possible with both ligands. The results show that Mg(2+) and Mn(2+) exist as open (op) isomers binding to both dinucleotides only at the terminal phosphate group. Whereas Cd(pUpU)(-) only exists as Cd(pUpU)(-)(op), Cd(pUp((S))U)(-) is present to about 64 % as the S-coordinated macrochelate, Cd(pUp((S))U)(-)(cl/PS). Zn(2+) forms with pUp((S))U(3-) three isomeric species, that is, Zn(pUp((S))U)(-)(op), Zn(pUp((S))U)(-)(cl/PO), and Zn(pUp((S))U)(-)(cl/PS), which occur to about 33, 12 (O-bound), and 55 %, respectively. Pb(2+) forms the 10-membered chelate with both nucleotides involving only the terminal oxygen atoms of the (thio)phosphate bridge, that is, no indication of S binding was discovered in this case. Hence, Zn(2+) and Cd(2+) show pronounced thiophilic properties, whereas Mg(2+), Mn(2+), and Pb(2+) coordinate to the oxygen, macrochelate formation being of relevance with Pb(2+) only.

Multisignaling detection of Hg2+ based on a phosphorescent iridium(iii) complex

A multisignaling chemosensor for Hg(2+) based on the iridium(III) complex Ir(thq)(2)(acac) was realized through UV-Vis absorption, phosphorescent emission and electrochemical measurements. Upon addition of Hg(2+), an obvious blue-shift in absorption spectra and a strong decrease of emission intensity were measured for Ir(thq)(2)(acac), which could be observed by the naked eye. Hg(2+) is coordinated to Ir(thq)(2)(acac), forming a 1 : 1 complex. Because Hg(2+) is a thiophilic metal ion, the interaction between Hg(2+) and the sulfur atom of cyclometalated ligands is responsible for the significant variations in optical and electrochemical signals.

Modulation of individual steps in group I intron catalysis by a peripheral metal ion

Enzymes are complex macromolecules that catalyze chemical reactions at their active sites. Important information about catalytic interactions is commonly gathered by perturbation or mutation of active site residues that directly contact substrates. However, active sites are engaged in intricate networks of interactions within the overall structure of the macromolecule, and there is a growing body of evidence about the importance of peripheral interactions in the precise structural organization of the active site. Here, we use functional studies, in conjunction with published structural information, to determine the effect of perturbation of a peripheral metal ion binding site on catalysis in a well-characterized catalytic RNA, the Tetrahymena thermophila group I ribozyme. We perturbed the metal ion binding site by site-specifically introducing a phosphorothioate substitution in the ribozyme's backbone, replacing the native ligands (the pro-R (P) oxygen atoms at positions 307 and 308) with sulfur atoms. Our data reveal that these perturbations affect several reaction steps, including the chemical step, despite the absence of direct contacts of this metal ion with the atoms involved in the chemical transformation. As structural probing with hydroxyl radicals did not reveal significant change in the three-dimensional structure upon phosphorothioate substitution, the effects are likely transmitted through local, rather subtle conformational rearrangements. Addition of Cd(2+), a thiophilic metal ion, rescues some reaction steps but has deleterious effects on other steps. These results suggest that native interactions in the active site may have been aligned by the naturally occurring peripheral residues and interactions to optimize the overall catalytic cycle.

A General RNA-Capping Ribozyme Retains Stereochemistry during Cap Exchange

Numerous natural and artificial ribozymes have been shown to facilitate reactions that invert stereochemistry. Here, we demonstrate that an RNA-capping ribozyme retains stereochemistry at a phosphorus reaction center. The ribozyme synthesizes a broad range of 5'-5' RNA caps by exchanging phosphate groups around the alpha-phosphate found at the 5' terminus of the ribozyme. A ribozyme prepared with an Rp adenosine(5')alpha-thiotetraphosphate cap was found to exchange this cap for an Rp 4-thiouridine(5')alpha-thiotetraphosphate cap when incubated with 4-thiouridine triphosphate. The same Rp capped construct, when incubated with [gamma-(32)P]-ATP, exchanged the unlabeled ATP for a radiolabeled one while maintaining the same stereoconfiguration. In contrast, ribozymes prepared with an Sp cap failed to react even in the presence of thiophilic metal ions such as manganese. The kinetics of capping was also unusual as compared to inverting ribozymes. When the ribozyme was prepared with a triphosphate, capping was found to follow Michaelis-Menten-type kinetics even though the rate of pyrophosphate release was completely independent of nucleotide substrate concentration. Interestingly, the rate of capping and hydrolysis, when summed, was found to be indistinguishable from the rate of pyrophosphate release, indicating that an early rate-limiting step precedes both capping and hydrolysis. Together the retention of stereochemistry and kinetics imply that capping utilizes two inverting chemical steps that are separated by the transient formation of a rate-limiting covalent intermediate. As all protein enzymes that mediate similar capping reactions utilize a covalent intermediate, chemical necessity may have strongly guided the evolution of both protein and RNA-capping catalysts.

Metal Clusters as Ligands. Substitution of Fe Ions in Fe/Mo/S Clusters by Thiophilic CuI Ions

The reactivity of Fe/S and Fe/Mo/S clusters, similar or analogous to those occurring in biological systems, with thiophilic metal ions has not been explored. In this Communication, we demonstrate that synthesis of heteropolynuclear clusters with different coordination geometries for different metals at different sites is possible by metal substitution or by metal addition reactions. The two clusters we report herein ([(Cl4-cat)2Mo2Cu5Fe4S9(PnPr3)7(SPnPr3)2]PF6 and [(Cl4-cat)2Mo2Cu6Fe4S10(PnPr3)8]) contain Fe, Mo, and Cu, which display pseudotetrahedral, pseudooctahedral, and pseudotrigonal geometries, respectively. The synthesis of these clusters is achieved by the addition of appropriate amounts of [Cu(CH3CN)4]+ to [(Cl4-cat)2Mo2Fe8(PnPr3)6]. The formation of the different products is temperature- and solvent-dependent. The Cu(I) units incorporated into the metal cluster framework, either bind to available lone pairs of the already bridging S ligands or displace the less thiophilic Fe atoms. Among the essential features of these new molecules are recognizable Fe/S fragments including an Fe6S9 core in the first cluster and the pentlandite Fe4Cu4S6 core in the second cluster.

Engineered Single-Chain, Antiparallel, Coiled CoilMimics the MerR Metal BindingSite

The repressor-activator MerR that controls transcription of the mercury resistance (mer) operon is unusual for its high sensitivity and specificity for Hg(II) in in vivo and in vitro transcriptional assays. The metal-recognition domain of MerR resides at the homodimer interface in a novel antiparallel arrangement of alpha-helix 5 that forms a coiled-coil motif. To facilitate the study of this novel metal binding motif, we assembled this antiparallel coiled coil into a single chain by directly fusing two copies of the 48-residue alpha-helix 5 of MerR. The resulting 107-residue polypeptide, called the metal binding domain (MBD), and wild-type MerR were overproduced and purified, and their metal-binding properties were determined in vivo and in vitro. In vitro MBD bound ca. 1.0 equivalent of Hg(II) per pair of binding sites, just as MerR does, and it showed only a slightly lower affinity for Hg(II) than did MerR. Extended X-ray absorption fine structure data showed that MBD has essentially the same Hg(II) coordination environment as MerR. In vivo, cells overexpressing MBD accumulated 70 to 100% more (203)Hg(II) than cells bearing the vector alone, without deleterious effects on cell growth. Both MerR and MBD variously bound other thiophilic metal ions, including Cd(II), Zn(II), Pb(II), and As(III), in vitro and in vivo. We conclude that (i) it is possible to simulate in a single polypeptide chain the in vitro and in vivo metal-binding ability of dimeric, full-length MerR and (ii) MerR's specificity in transcriptional activation does not reside solely in the metal-binding step.

Evidence for a polynuclear metal ion binding site in the catalytic domain of ribonuclease P RNA.

Interactions with divalent metal ions are essential for the folding and function of the catalytic RNA component of the tRNA processing enzyme ribonuclease P (RNase P RNA). However, the number and location of specific metal ion interactions in this large, highly structured RNA are poorly understood. Using atomic mutagenesis and quantitative analysis of thiophilic metal ion rescue we provide evidence for metal ion interactions at the pro-R(P) and pro-S(P) non-bridging phosphate oxygens at nucleotide A67 in the universally conserved helix P4. Moreover, second-site modifications within helix P4 and the adjacent single stranded region (J3/4) provide the first evidence for metal ion interactions with nucleotide base functional groups in RNase P RNA and reveal the presence of an additional metal ion important for catalytic function. Together, these data are consistent with a cluster of metal ion interactions in the P1-P4 multi-helix junction that defines the catalytic core of the RNase P ribozyme.

Identification of an active site ligand for a group I ribozyme catalytic metal ion.

The transition state of the group I intron self-splicing reaction is stabilized by three metal ions. The functional groups within the intron substrates (guanosine and an oligoribonucleotide mimic of the 5'-exon) that coordinate these metal ions have been systematically defined through a series of metal ion specificity switch experiments. In contrast, the catalytic metal ligands within the ribozyme active site are unknown. In an effort to identify them, stereospecific (R(P) or S(P)) single-site phosphorothioate substitutions were introduced at five phosphates predicted to be in the vicinity of the catalytic center (A207, C208, A304, U305, and A306) within the Tetrahymena intron. Of the 10 ribozymes that were studied, four phosphorothioate substitutions (A207 S(P), C208 S(P), A306 R(P), and A306 S(P)) exhibited a significant reduction in the cleavage rate. Only the effect of the C208 S(P) phosphorothioate substitution could be significantly rescued by the addition of a thiophilic metal ion, either Mn(2+) or Zn(2+), when tested with an all-oxy substrate. The effect was not rescued with Cd(2+). To determine if one of the catalytic metal ions is coordinated to the C208 pro-S(P) oxygen, the phosphorothioate-substituted ribozymes were also assayed using oligonucleotide substrates with a 3'-phosphorothiolate or an S(P) phosphorothioate substitution at the scissile phosphate. This resulted in a second metal specificity switch, in that Mn(2+) or Zn(2+) no longer rescued the C208 S(P) ribozyme, but Cd(2+) provided efficient rescue in the context of either sulfur-containing substrate. The 3'-oxygen and the pro-S(P) oxygen of the scissile phosphate are both known to coordinate the same metal ion, M(A), which stabilizes the negative charge on the leaving group 3'-oxygen in the transition state. Taken together, these data suggest that metal M(A) is coordinated to the C208 pro-S(P) phosphate oxygen, which constitutes the first functional link between a specific catalytic metal ion and a particular functional group within the group I ribozyme active site.