Lead Ion Binding and RNA Chain Hydrolysis in Phenylalanine tRNA

Abstract

Crystalline complexes of yeast phenylalanine tRNA and Lead (II) ion were prepared by soaking pregrown orthorhombic crystals of tRNA in saturated lead chloride solutions. The locations of tightly bound lead ions on the tRNA were determined by difference Fourier methods. There are three major lead binding sites; two of these replace tightly bound magnesium ions in the native tRNA structure. Site I is located in the dihydrouridine loop of the molecule adjacent to phosphate P18 which is specifically cleaved by lead. This is evident from changes observed in the Pb-native difference electron density maps. A possible mechanism for lead ion hydrolysis of the polynucleotide chain is proposed.     

Differential association of transfer RNAs with the genomes of murine, feline and primate retroviruses

The tRNAs that are bound to the genomic RNAs of several murine, feline, and primate retroviruses have been identified. Transfer RNAs were divided into those loosely bound and those tightly bound by stepwise thermal dissociation of the 70 S RNA. They were then identified and semiquantitated by aminoacylation. Proline tRNA is the most tenaciously bound tRNA in several strains of murine leukemia virus, two strains of feline leukemia virus, and the primate viruses simian sarcoma, baboon endogenous, and gibbon ape lymphoma. In the feline xenotropic virus, RD-114, tRNAGly is enriched in the most tightly bound fraction. In Mason-Pfizer monkey virus, as in the murine mammary tumor virus, tRNALys is the tRNA most tenaciously bound to its genomic RNA. Besides the most tightly associated tRNA, one or more different tRNAs are found in relatively large amounts in association with the 70 S RNA. (For convenience, we refer to the largest RNA ccomplex (50-70 S) isolated from any of the retroviruses studies as '70 S' RNA.) These tRNAs can be distinguished from the most tightly bound tRNA by the fact that they can be dissociated at lower temperatures. However, they occur in the same relative abundance as the tightly bound tRNA.     

Predicting RNA-Metal Ion Binding with Ion Dehydration Effects

Metal ions play essential roles in nucleic acids folding and stability. The interaction between metal ions and nucleic acids can be highly complicated because of the interplay between various effects such as ion correlation, fluctuation, and dehydration. These effects may be particularly important for multivalent ions such as Mg²⁺ ions. Previous efforts to model ion correlation and fluctuation effects led to the development of the Monte Carlo tightly bound ion model. Here, by incorporating ion hydration/dehydration effects into the Monte Carlo tightly bound ion model, we develop a, to our knowledge, new approach to predict ion binding. The new model enables predictions for not only the number of bound ions but also the three-dimensional spatial distribution of the bound ions. Furthermore, the new model reveals several intriguing features for the bound ions such as the mutual enhancement/inhibition in ion binding between the fully hydrated (diffuse) ions, the outer-shell dehydrated ions, and the inner-shell dehydrated ions and novel features for the monovalent-divalent ion interplay due to the hydration effect.     

Dynamic hydration numbers for biologically important ions

The role of ionized groups in biological systems is determined by their affinity for water [Biophys. J. 72 (1997) 65-76]. The tightly bound water associated with biologically important ions increases their apparent size. We define the apparent dynamic hydration number of an ion here as the number of tightly bound water molecules that must be assigned to the ion to explain its apparent molecular weight on a Sephadex G-10 size exclusion column, and report the first accurate determination of tightly bound water for 23 ions of biological significance, including H(+) and HO(-). We also calculate the radius of the equivalent hydrated sphere (r(h)) for each ion. We find that the ratio of the hydrated volumes of two ions approximates the ratio of the square of the charges of the same two ions. Since the 'ionic strength' of the solution also depends upon the square of the charges on the ions, our results suggest that ionic strength effects may largely arise from local effects related to the hydrated volume of the ion--that is, from space filling, osmotic, water activity, surface tension and hydration shell overlap effects rather than from long-range electric field effects.     

The crystal structure of yeast phenylalanine tRNA at 2.0 A resolution: cleavage by Mg(2+) in 15-year old crystals

We have re-determined the crystal structure of yeast tRNA(Phe) to 2. 0 A resolution using 15 year old crystals. The accuracy of the new structure, due both to higher resolution data and formerly unavailable refinement methods, consolidates the previous structural information, but also reveals novel details. In particular, the water structure around the tightly bound Mg(2+) is now clearly resolved, and hence provides more accurate information on the geometry of the magnesium-binding sites and the role of water molecules in coordinating the metal ions to the tRNA. We have assigned a total of ten magnesium ions and identified a partly conserved geometry for high-affinity Mg(2+ )binding. In the electron density map there is also clear density for a spermine molecule binding in the major groove of the TPsiC arm and also contacting a symmetry-related tRNA molecule. Interestingly, we have also found that two specific regions of the tRNA in the crystals are partially cleaved. The sites of hydrolysis are within the D and anticodon loops in the vicinity of Mg(2+).     

RNA helix stability in mixed Na+/Mg2+ solution

A recently developed tightly bound ion model can account for the correlation and fluctuation (i.e., different binding modes) of bound ions. However, the model cannot treat mixed ion solutions, which are physiologically relevant and biologically significant, and the model was based on B-DNA helices and thus cannot directly treat RNA helices. In the present study, we investigate the effects of ion correlation and fluctuation on the thermodynamic stability of finite length RNA helices immersed in a mixed solution of monovalent and divalent ions. Experimental comparisons demonstrate that the model gives improved predictions over the Poisson-Boltzmann theory, which has been found to underestimate the roles of multivalent ions such as Mg2+ in stabilizing DNA and RNA helices. The tightly bound ion model makes quantitative predictions on how the Na+-Mg2+ competition determines helix stability and its helix length-dependence. In addition, the model gives empirical formulas for the thermodynamic parameters as functions of Na+/Mg2+ concentrations and helix length. Such formulas can be quite useful for practical applications.     

Role of divalent ions in folding of tRNA.

The native structure of tRNA is not achieved in low salt (4.5 mM Na+, 25 degrees C), but can be restored by addition of divalent ions. We have explored the structure of the central region in Escherichia coli tRNAfMet by absorption and emission spectroscopy of 4-thiouracil, and the structure of the anticodon loop in yeast tRNAPhe by fluorescence of the 'Y' base, versus the number of manganese ions bound to tRNA, which was derived from electron spin resonance. The fluorescence of the reduced 8-13 photoproduct (in which 4-thiouracil at position 8 is crosslinked to cytosine at position 13) was also analysed. In low salt (e.g. 4.5 mM Na+), the region of 4-thiouracil is affected strongly as the first eight Mn2+ bind to tRNA, whereas the fluorescence of the 'Y' base is affected only after four Mn2+ are bound. Considering the structural similarities of the two tRNAs, this suggests that the reorganisation brought about by divalent ions starts in the central region, the anticodon loop being affected later. The binding of divalent ions to each region starts together with its restructuration. Monovalent ions can substitute for divalent ions in this process, a 15 mM sodium concentration being equivalent to the binding of the first five Mn2+. If divalent ions are then added, even the first ones distribute themselves between both the central and the anticodon region. Alternatively, the renaturation may be achieved by monovalent ions only, implying that no sites exist whose occupancy by divalent ions is crucial for the native structure. These observations suggest that the role and means of divalent ion binding to tRNA are largely explainable in terms of a simple maganese-phosphate binding supplemented by electrostatic interaction with distant phosphates.     

Characterization of the lead(II)-induced cleavages in tRNAs in solution and effect of the Y-base removal in yeast tRNAPhe

The specificity of lead(II)-induced hydrolysis of yeast tRNA(Phe) was studied as a function of concentration of Pb2+ ions. The major cut was localized in the D-loop and minor cleavages were detected in the anticodon and T-loops at high metal ion concentration. The effects of pH, temperature, and urea were also analyzed, revealing a basically unchanged specificity of hydrolysis. In the isolated 5'-half-molecule of yeast tRNAPhe not cut was found in the D-loop, indicating its stringent dependence on T-D-loop interaction. Comparison of hydrolysis patterns and efficiencies observed in yeast tRNA(Phe) with those found in other tRNAs suggests that the presence of a U59-C60 sequence in the T-loop is responsible for the highly efficient and specific hydrolysis in the spatially close region of the D-loop. The efficiencies of D-loop cleavage in intact yeast tRNA(Phe) and in tRNA(Phe) deprived of the Y base next to the anticodon were also compared at various Pb2+ ion concentrations. Kinetics of the D-loop hydrolysis analyzed at 0, 25, and 37 degrees C showed a 6 times higher susceptibility of tRNA(Phe) minus Y base (tRNA(Phe)-Y) to lead(II)-induced hydrolysis than in tRNA(Phe). The observed effect is discussed in terms of a long-distance conformational transition in the region of the interacting D- and T-loops triggered by the Y-base excision.     

Effect of europium(III) on the thermal denaturation and cleavage of transfer ribonucleic acids

The interaction of tRNA with Eu(III) has been studied by optical and gel electrophoretic tecfhniques. At low levels (less than six metals per tRNA), Eu(III) stabilizes yeast tRNA^Phe against thermal denaturation; however, at higher levels (about eight to ten Eu/tRNA) the tRNA is destabilized by Eu(III). This may have important implications regarding recent attempts to grow crystals of tRNA from solutions containing europium. Comparative studies of the effects of Mg(II) and Eu(III) on tRNA structure confirm that the first four Eu(III) ions are more strongly bound than Mg(II). At slightly elevated temperatures (50°C, pH 7) the binding of Eu(III) catalyzes the hydrolysis of the tRNA backbone. From an analysis of the fragments produced by the hydrolysis and of the variation in the rate of cleavage as a function of the metal per tRNA ratio, we conclude that (i) the addition of Eu(III) to the tRNA is sequential, (ii) the first Eu(III) is bound in close proximity to the two dihydrouridine residues, and (iii) the rate of hydrolysis depends on the number of Eu(III) free in solution. Metals bound at sites 2–4 are relatively much less active in promoting the cleavage but the metal bound at site 5 is again active. The initial cleavage products are shown to be identical with those obtained using magnesium, zinc, or lead.     

Ion dependence of the Bacillus subtilis RNase P reaction

The properties of the Bacillus subtilis RNase P are characterized with regard to the types and concentrations of monovalent and divalent ions required to potentiate precursor tRNA cleavage by the protein-RNA holoenzyme and the catalytic RNA alone. The ionic dependence of the RNase P RNA-catalyzed reaction in part seems due to a requirement for ion shielding between substrate and catalytic RNAs. The RNase P protein, which binds to RNA nonspecifically and tightly, likely serves, in part, as a cation screen. However, the character of the ion dependence of the RNA catalysis, the inhibition by high SO2-4 concentration, and potentiation by solvents suggest that RNA conformational transition may be involved in the reaction. It is proposed that the reason for catalysis by RNA in the RNase P reaction may be a requirement for fluidity in the structure of the catalyst, so that it can accommodate many tRNA substrates, which vary in their structural details.     

Evidence against bicarbonate bound in the O2-evolving complex of photosystem II

The oxidation of water to molecular oxygen by photosystem II (PSII) is inhibited in bicarbonate-depleted media. One contribution to the inhibition is the binding of bicarbonate to the non-heme iron, which is required for efficient electron transfer on the electron-acceptor side of PSII. There are also proposals that bicarbonate is required for formation of O 2 by the manganese-containing O 2-evolving complex (OEC). Previous work indicates that a bicarbonate ion does not bind reversibly close to the OEC, but it remains possible that bicarbonate is bound sufficiently tightly to the OEC that it cannot readily exchange with bicarbonate in solution. In this study, we have used NH 2OH to destroy the OEC, which would release any tightly bound bicarbonate ions from the active site, and mass spectrometry to detect any released bicarbonate as CO 2. The amount of CO 2 per PSII released by the NH 2OH treatment is observed to be comparable to the background level, although N 2O, a product of the reaction of NH 2OH with the OEC, is detected in good yield. These results strongly argue against tightly bound bicarbonate ions in the OEC.     

Roles of metal ions in the maintenance of the tertiary and quaternary structure of arginase from Saccharomyces cerevisiae

Arginase from Saccharomyces cerevisiae has long been known to be a metal ion-requiring enzyme as it requires heating at 45 degrees C in the presence of 10 mM Mn2+ for catalytic activation. Metals are also thought to play a structural role in the enzyme, but the identity of the structural metal and its precise structural role have not been defined. Analysis of the metal ions that bind to yeast arginase by atomic absorption spectroscopy reveals that there is a weakly associated Mn2+ that binds to the trimeric enzyme with a stoichiometry of 1.04 +/- 0.05 mol of Mn2+ bound per subunit and an apparent K'D value of 26 microM at pH 7.0 and 4 degrees C. A more tightly associated Zn2+ ion can only be removed by dialysis against chelating agents. In occasional preparations, this site contained some Mn2+; however, Zn2+ and Mn2+ together bind to high affinity sites with a stoichiometry of 1.14 +/- 0.25/mol of subunit. Both the loosely associated catalytic Mn2+ ion and the more tightly associated structural Zn2+ ion confer stability to the enzyme. Removal of the weakly bound Mn2+ ion results in a 3 degree C decrease in the midpoint of the thermal transition (T 1/2) (from 57 by 54 degrees C) as monitored by UV difference absorption spectroscopy. Removal of the tightly bound Zn2+ ion produces a 19 degrees C decrease in T 1/2 (to 38 degrees C). Similar results are obtained by circular dichroism measurements. When the Zn2+ ion is removed, the steady-state fluorescence intensity increases 100% as compared to the holoenzyme, with a shift in the emission maximum from 337 to 352 nm. This suggests that in the folded trimeric metalloenzyme, the tryptophan fluorescence is quenched and that upon removal of the structural metal, the quenching is relieved as tryptophan residues become exposed to more polar environments. Equilibrium sedimentation experiments performed after dialysis of the enzyme against EDTA demonstrate that arginase exists in a reversible monomer-trimer equilibrium, in the absence of metal ions, with a KD value of 5.05 x 10(-11) M2. In contrast, the native enzyme exists as a trimer with no evidence of dissociation when Mn2+ and Zn2+ are present (Eisenstein, E., Duong, L.T., Ornberg, R. L., Osborne, J.C., Jr., and Hensley, P. (1986) J. Biol. Chem. 261, 12814-12819). In summary, the study presented here demonstrates that binding of a weakly bound Mn2+ ion confers catalytic activity. In contrast, binding of a more tightly associated Zn2+ ion confers substantial stability to the tertiary and quaternary structure of the enzyme.     

On the coordination properties of Eu3+ bound to tRNA

The luminescence properties of Eu3+ have been used to investigate the binding and coordination properties of the ion with tRNA, as an attempt to resolve the discussion of whether metal ions bind to tRNA in solution only by Debye-Hückel screening, or whether direct coordination to specific sites may occur. Binding studies with Escherichia coli tRNAmet/f (taking advantage of 4-thiouracil-sensitized Eu3+ emission) distinguish three classes of binding affinities. Two of these are single sites with affinities approx. 10(4) and approx. 10(3) tighter than the nonspecific affinity of Eu3+ for native DNA. Mg2+ competes for binding at both these sites. Measurement of the lifetime and excitation spectrum of Eu3+ bound to the highest affinity site shows that the ion has two to five non-phosphate ligands in its inner coordination sphere. The existence of this coordinated site demonstrates that electrostatic screening is not the only mechanism for metal ion interaction with tRNA. The coordination properties of the high-affinity Eu3+ site do not agree with the properties of any of the metal ion sites found in the two tRNAphe crystal forms. Possible reasons for this discrepancy are discussed; it may be that ions bind differently to isolated molecules in solution than to molecules packed in a crystal lattice.     

Binding of the Activating Ion Co(II) to myo-Inositol Monophosphatase Monitored by Fluorescence and Phosphorescence Spectroscopy

Two extrinsic probes, pyrene-maleimide and eosin-maleimide, were used to label specific SH groups of the enzyme myo-inositol monophosphatase. The fluorescence of pyrene-monophosphatase is enhanced upon addition of the activating metal ions Co(II) and Mg(II). Co(II) ions bind with a dissociation constant of 4 μM, whereas the apparent activation constant K _a is 0.4 mM. Energy transfer measurements demonstrated that the pyrene chromophore, covalently linked to Cys-218, is within 9 Å of the metal ion Tb(III) coordinated to the metal-binding site. The phosphorescence emitted by eosin covalently linked to the protein is quenched by the addition of the activating cations Co(II) and Mg(II). Phosphorescence titrations conducted under anaerobic conditions were used to determine a dissociation constant of approximately 3 μM for the binding of Co(II) ions. The results are consistent with the hypothesis that two activating ions per monomeric subunit participate in the catalytic mechanism. The affinity of the tightly bound ion is at least 100-fold greater than the affinity of the weakly bound ion.     

Reinvestigation of metal ion specificity for quinone cofactor biogenesis in bacterial copper amine oxidase

The topa quinone (TPQ) cofactor of copper amine oxidase is generated by copper-assisted self-processing of the precursor protein. Metal ion specificity for TPQ biogenesis has been reinvestigated with the recombinant phenylethylamine oxidase from Arthrobacter globiformis. Besides Cu2+ ion, some divalent metal ions such as Co2+, Ni2+, and Zn2+ were also bound to the metal site of the apoenzyme so tightly that they were not replaced by excess Cu2+ ions added subsequently. Although these noncupric metal ions could not initiate TPQ formation under the atmospheric conditions, we observed slow spectral changes in the enzyme bound with Co2+ or Ni2+ ion under the dioxygen-saturating conditions. Resonance Raman spectroscopy and titration with phenylhydrazine provided unambiguous evidence for TPQ formation by Co2+ and Ni2+ ions. Steady-state kinetic analysis showed that the enzymes activated by Co2+ and Ni2+ ions were indistinguishable from the corresponding metal-substituted enzymes prepared from the native copper enzyme (Kishishita, S., Okajima, T., Kim, M., Yamaguchi, H., Hirota, S., Suzuki, S., Kuroda, S., Tanizawa, K., and Mure, M. (2003) J. Am. Chem. Soc. 125, 1041-1055). X-ray crystallographic analysis has also revealed structural identity of the active sites of Co- and Ni-activated enzymes with Cu-enzyme. Thus Cu2+ ion is not the sole metal ion assisting TPQ formation. Co2+ and Ni2+ ions are also capable of forming TPQ, though much less efficiently than Cu2+.     

Quantitative analysis of the ion-dependent folding stability of DNA triplexes

A DNA triplex is formed through binding of a third strand to the major groove of a duplex. Due to the high charge density of a DNA triplex, metal ions are critical for its stability. We recently developed the tightly bound ion (TBI) model for ion-nucleic acids interactions. The model accounts for the potential correlation and fluctuations of the ion distribution. We now apply the TBI model to analyze the ion dependence of the thermodynamic stability for DNA triplexes. We focus on two experimentally studied systems: a 24-base DNA triplex and a pair of interacting 14-base triplexes. Our theoretical calculations for the number of bound ions indicate that the TBI model provides improved predictions for the number of bound ions than the classical Poisson-Boltzmann (PB) equation. The improvement is more significant for a triplex, which has a higher charge density than a duplex. This is possibly due to the higher ion concentration around the triplex and hence a stronger ion correlation effect for a triplex. In addition, our analysis for the free energy landscape for a pair of 14-mer triplexes immersed in an ionic solution shows that divalent ions could induce an attractive force between the triplexes. Furthermore, we investigate how the protonated cytosines in the triplexes affect the stability of the triplex helices.     

The calmodulin-dependent activation and deactivation of the phosphoprotein phosphatase, calcineurin, and the effect of nucleotides, pyrophosphate, and divalent metal ions. Identification of calcineurin as a Zn and Fe metalloenzyme

Studies on the interaction of calcineurin with its activator, calmodulin, showed that the 1:1 complex is the activated species. Concomitant with activation, a time-dependent deactivation of the phosphatase was observed. The process followed first order kinetics and was dependent on the presence of both Ca2+ and calmodulin. The deactivation rate constant at pH 7.6 and 30 degrees C was 0.06 min-1, which was increased by the substrate, p-nitrophenylphosphate (Km = 11 mM), to 0.47 min-1. PPi and nucleotides inhibited the enzyme competitively and accelerated the deactivation. The first order rate constant was increased to 2.3 min-1 by PPi (Ki = 55 microM) and to 8.0 min-1 by ADP (Ki = 0.94 mM). A theory dealing with the deactivation (applicable to chemical modification, etc.) of an enzyme in the absence and presence of various ligands is presented. The deactivated enzyme remained bound to calmodulin and was not reactivated by dissociation-reassociation of the calcineurin-calmodulin complex. Calcineurin was found to contain covalently bound phosphate; however, no difference in its content was detected upon deactivation, indicating that self-dephosphorylation was not involved. The deactivation could be reversed, as well as prevented, by divalent metal ions such as Ni2+ and Mn2+. Atomic absorption spectroscopy revealed nearly stoichiometric amounts of tightly bound Fe and Zn (but little other ions) on purified calcineurin, which remained bound during the calmodulin-dependent deactivation; removal of tightly bound metals is, therefore, not the cause of deactivation. Our results indicate that calcineurin is a metallophosphatase and not simply a Ca2+- and calmodulin-stimulated enzyme. In addition to the nondissociable Zn and Fe and the Ca2+ bound to the B subunit and calmodulin, the enzyme requires a divalent metal ion for structural stability and full activity.     

Tetracycline can inhibit tRNA binding to the ribosomal P site as well as to the A site

A and P sites of Escherichia coli ribosomes were titrated with AcPhe-tRNAPhe, in the absence or presence of tetracycline. The P-site location of the bound AcPhe-tRNA was assessed by means of a quantitative puromycin reaction. The results demonstrate that, in agreement with the generally held view, tetracycline exclusively inhibits the A-site binding, if the statistical number of bound acyl-tRNA molecules per ribosome does not exceed about 0.5. However, above this value the P site becomes sensitive to tetracycline as well. It follows that the tightly coupled 70S ribosomes used in functional studies appear to be functionally heterogeneous, i.e. those P sites which cannot be affected by tetracycline are preferentially occupied by AcPhe-tRNA, whereas higher concentrations of this tRNA species are required to fill tetracycline-sensitive P sites. Furthermore, the results imply that under tRNA saturation conditions the tetracycline inhibition cannot be used as an indicator for the site location of bound tRNA.