Mapping divalent metal ion binding sites in a group II intron by Mn(2+)- and Zn(2+)-induced site-specific RNA cleavage.

The function of group II introns depends on positively charged divalent metal ions that stabilize the ribozyme structure and may be directly involved in catalysis. We investigated Mn2+- and Zn2+-induced site-specific RNA cleavage to identify metal ions that fit into binding pockets within the structurally conserved bI1 group II intron domains (DI-DVI), which might fulfill essential roles in intron function. Ten cleavage sites were identified in DI, two sites in DIII and two in DVI. All cleavage sites are located in the center or close to single-stranded and flexible RNA structures. Strand scissions mediated by Mn2+/Zn2+ are competed for by Mg2+, indicating the existence of Mg2+ binding pockets in physical proximity to the observed Mn2+-/Zn2+-induced cleavage positions. To distinguish between metal ions with a role in structure stabilization and those that play a more specific and critical role in the catalytic process of intron splicing, we combined structural and functional assays, comparing wild-type precursor and multiple splicing-deficient mutants. We identified six regions with binding pockets for Mg2+ ions presumably playing an important role in bI1 structure stabilization. Remarkably, assays with DI deletions and branch point mutants revealed the existence of one Mg2+ binding pocket near the branching A, which is involved in first-step catalysis. This pocket formation depends on precise interaction between the branching nucleotide and the 5' splice site, but does not require exon-binding site 1/intron binding site 1 interaction. This Mg2+ ion might support the correct placing of the branching A into the 'first-step active site'.     

Lead cleavage sites in the core structure of group I intron-RNA.

Self-splicing of group I introns requires divalent metal ions to promote catalysis as well as for the correct folding of the RNA. Lead cleavage has been used to probe the intron RNA for divalent metal ion binding sites. In the conserved core of the intron, only two sites of Pb2+ cleavage have been detected, which are located close to the substrate binding sites in the junction J8/7 and at the bulged nucleotide in the P7 stem. Both lead cleavages can be inhibited by high concentrations of Mg2+ and Mn2+ ions, suggesting that they displace Pb2+ ions from the binding sites. The RNA is protected from lead cleavage by 2'-deoxyGTP, a competitive inhibitor of splicing. The two major lead induced cleavages are both located in the conserved core of the intron and at phosphates, which had independently been demonstrated to interact with magnesium ions and to be essential for splicing. Thus, we suggest that the conditions required for lead cleavage occur mainly at those sites, where divalent ions bind that are functionally involved in catalysis. We propose lead cleavage analysis of functional RNA to be a useful tool for mapping functional magnesium ion binding sites.     

Metal ion binding sites in a group II intron core

Group II introns are catalytic RNA molecules that require divalent metal ions for folding, substrate binding, and chemical catalysis. Metal ion binding sites in the group II core have now been elucidated by monitoring the site-specific RNA hydrolysis patterns of bound ions such as Tb(3+) and Mg(2+). Major sites are localized near active site elements such as domain 5 and its surrounding tertiary interaction partners. Numerous sites are also observed at intron substructures that are involved in binding and potentially activating the splice sites. These results highlight the locations of specific metal ions that are likely to play a role in ribozyme catalysis.     

Structural metals in the group I intron: a ribozyme with a multiple metal ion core

Metal ions play key roles in the folding and function for many structured RNAs, including group I introns. We determined the X-ray crystal structure of the Azoarcus bacterial group I intron in complex with its 5' and 3' exons. In addition to 222 nucleotides of RNA, the model includes 18 Mg(2+) and K(+) ions. Five of the metals bind within 12 A of the scissile phosphate and coordinate the majority of the oxygen atoms biochemically implicated in conserved metal-RNA interactions. The metals are buried deep within the structure and form a multiple metal ion core that is critical to group I intron structure and function. Eight metal ions bind in other conserved regions of the intron structure, and the remaining five interact with peripheral structural elements. Each of the 18 metals mediates tertiary interactions, facilitates local bends in the sugar-phosphate backbone or binds in the major groove of helices. The group I intron has a rich history of biochemical efforts aimed to identify RNA-metal ion interactions. The structural data are correlated to the biochemical results to further understand the role of metal ions in group I intron structure and function.     

Structural requirement for Mg2+ binding in the group I intron core

Divalent metal ions are required for splicing of group I introns, but their role in maintaining the structure of the active site is still under investigation. Ribonuclease and hydroxyl radical footprinting of a small group I intron from Azoarcus pre-tRNA(Ile) showed that tertiary interactions between helical domains are stable in a variety of cations. Only Mg(2+), however, induced a conformational change in the intron core that correlates with self-splicing activity. Three metal ion binding sites in the catalytic core were identified by Tb(III)-dependent cleavage. Two of these are near bound substrates in a three-dimensional model of the ribozyme. A third metal ion site is near an A minor motif in P3. In the pre-tRNA, Tb(3+) cleavage was redirected to the 5' and 3' splice sites, consistent with metal-dependent activation of splice site phosphodiesters. The results show that many counterions induce global folding, but organization of the group I active site is specifically linked to Mg(2+) binding at a few sites.     

Specificity of Mg2+ binding at the Group II intron branch site

Metal ions play a crucial role in the conformation and splicing activity of Group II introns. Results from 2-aminopurine fluorescence and solution NMR studies suggest that metal ion binding within the branch site region of native D6 of the Group II intron is specific for alkaline earth metal ions and involves inner sphere coordination. Although Mg(2+) and Ca(2+) still bind to a mutant stem loop sequence from which the internal loop had been deleted, ion binding to the mutant RNA results in decreased, rather than increased, exposure of the branch site residue to solvent. These data further support the role of the internal loop in defining branch site conformation of the Group II intron. The specific bound Mg(2+) may play a bivalent role: facilitates the extrahelical conformation of the branch site and has the potential to act as a Lewis acid during splicing.     

The binding of Mg(II) and Ni(II) to synthetic polynucleotides

Equilibria and kinetics of the interactions of Mg2+ and Ni2+ with poly(U), poly(C) and poly(I) have been investigated at 25 degrees C, an ionic strength of 0.1 M, and pH 7.0 or 6.0. Analogous studies involving poly(A) were reported earlier. All binding equilibria were studied by means of the (usually small) absorbance changes in the ultraviolet range. This technique yields apparent binding constants which are fairly large for the interaction of Ni2+ with poly(A) (K = 0.9 X 10(4) M-1) and poly(I) (K approximately equal to 2 X 10(4) M-1) but considerably lower for the corresponding Mg2+ systems, Mg2+-poly(A) (K = 2 X 10(3) M-1) and Mg2+-poly(I) (K = 280 M-1). Each of the two pyrimidine nucleotides binds both metal ions with about the same strength (K approximately equal to 65 M-1 for poly(U) and K near 600 M-1 for poly(C]. In the case of poly(C) the spectral changes deviate from those expected for a simple binding equilibrium. In addition, the binding of Ni2+ to the four polynucleotides was measured by using murexide as an indicator of the concentration of free Ni2+. The results obtained by this technique agree or are at least consistent with those derived from the ultraviolet spectra. Complications are encountered in the binding studies involving poly(I), particularly at higher metal ion concentrations, obviously due to the formation of aggregated poly(I) species. Kinetic studies of the binding processes were carried out by the temperature-jump relaxation technique. Measurable relaxation effects of time constants greater than 5 microseconds were observed only in the systems Ni2+-poly(A) and Ni2+-poly(I). Such not-too-fast reaction effects are expected for processes which include inner-sphere substitution steps at Mg2+ or Ni2+. The relaxation process in Ni2+-poly(I) is characterized by (at least) four time constants. Obviously, the complicated kinetics again include reactions of aggregated poly(I). The absence of detectable relaxation effects in all other systems (except Mg2+-poly(I), the kinetics of which was not investigated) indicates that inner-sphere coordination of the metal ions to specific sites of the polynucleotides (site binding) does not occur to a significant extent. Rather, the metal ions are bound in these systems mainly by electrostatic forces, forming a mobile cloud. The differences in binding strength which are nevertheless observed are attributed to differences in the conformation of the polynucleotides which result in different charge densities.     

Metal ion requirements for sequence-specific endoribonuclease activity of the Tetrahymena ribozyme

A shortened form of the self-splicing intervening sequence RNA of Tetrahymena thermophila acts as an enzyme, catalyzing sequence-specific cleavage of RNA substrates. We have now examined the metal ion requirements of this reaction. Mg2+ and Mn2+ are the only metal ions that by themselves give RNA enzyme activity. Atomic absorption spectroscopy indicates that Zn, Cu, Co, and Fe are not present in amounts equimolar to the RNA enzyme and when added to reaction mixtures do not facilitate cleavage. Thus, these ions can be eliminated as cofactors for the reaction. While Ca2+ has no activity by itself, it alleviates a portion of the Mg2+ requirement; 1 mM Ca2+ reduces the Mg2+ optimum from 2 to 1 mM. These results, combined with studies of the reactivity of mixtures of metal ions, lead us to postulate that two classes of metal ion binding sites are required for catalysis. Class 1 sites have more activity with Mn2+ than with Mg2+, with the other divalent ions and Na+ and K+ having no activity. It is not known if ions located at class 1 sites have specific structural roles or are directly involved in active-site chemistry. Class 2 sites, which are presumably structural, have an order of preference Mg2+ greater than or equal to Ca2+ greater than Mn2+ and Ca2+ greater than Sr2+ greater than Ba2+, with Zn2+, Cu2+, Co2+, Na+, and K+ giving no detectable activity over the concentration range tested.     

Solution structure of Cobalt(III)hexammine complexed to the GAAA tetraloop, and metal-ion binding to G.A mismatches

The solution structure of a 22 nt RNA hairpin and its complex with Co(NH(3))(6)(3+) bound to the GAAA tetraloop has been determined by NMR spectroscopy. Co(NH(3))(6)(3+) has a similar geometry to Mg(H(2)O)(6)(2+) and can be used as a probe for binding sites of completely solvated magnesium ions. The hairpin contains tandem G.A mismatches, similar to the P5abc region of a group I intron, and is closed by a GAAA tetraloop. The tandem G.A mismatches are imino hydrogen bonded in contrast with the sheared G.A mismatches found in a different context in the crystal structure of the P4-P6 domain. Chemical shift changes of the imino protons upon titration of the RNA hairpin with Mg(2+) and with Co(NH(3))(6)(3+) were used to identify ion-binding sites. Paramagnetic resonance broadening upon titration with Mn(2+) was also used. The titration curves gave dissociation binding constants for the magnesium ions in the millimolar range, similar to the binding in the major groove of RNA at tandem G.U base-pairs. Although the largest chemical shift change occurred at an imino proton of one of the G.A base-pairs, no nuclear Overhauser enhancement cross-peaks between the cobalt ligand and neighboring RNA protons were seen, presumably due to the high mobility of the Co(NH(3))(6)(3+) at this site. Nuclear Overhauser enhancement cross-peaks between Co(NH(3))(6)(3+) and the GAAA tetraloop were observed, which allowed the determination of the structure of the tetraloop binding site. The Co(NH(3))(6)(3+) is bound in the major groove of the GAAA tetraloop with hydrogen bonds to guanine base N7 and to phosphate oxygen atoms of the tetraloop.     

Evaluation of uranyl photocleavage as a probe to monitor ion binding and flexibility in RNAs

In order to evaluate uranyl photocleavage as a tool to identify and characterize structural and dynamic properties in RNA, we compared uranyl cleavage sites in five RNA molecules with known X-ray structures, namely the hammerhead and hepatitis delta virus ribozymes, the P4-P6 domain of the Tetrahymena group I intron, as well as tRNA(Phe) and tRNA(Asp) from yeast. Uranyl photocleavage was observed at specific positions in all molecules investigated. In order to characterize the sites, photocleavage was performed in the absence and in increasing amounts of MgCl(2). Uranyl photocleavage correlates well with sites of low calculated accessibility, suggesting that uranyl ions bind in tight RNA pockets formed by close approach of phosphate groups. RNA foldings require ion binding, usually magnesium ions. Thus, upon the adoption of the native structure, uranyl ions can no longer bind well except in flexible and open to the solvent regions that can undergo induced-fit without disrupting the native fold. Uranyl photocleavage was compared to N-ethyl-N-nitrosourea and lead-induced cleavages in the context of the three-dimensional X-ray structures. Overall, the regions protected from ENU attack are sites of uranyl cleavage, indicating sites of low accessibility which can form ion binding sites. On the contrary, lead cleavages occur at flexible and accessible sites and correlate with the unspecific cleavages prevalent in dynamic and open regions. Applied in a magnesium-dependent manner, and only in combination with other backbone probing agents such as N-ethyl-N-nitrosourea, lead and Fenton cleavage, uranyl probing has the potential to reveal high-affinity metal ion environments, as well as regions involved in conformational transitions.     

Functional characterization of lanthanide binding sites in the sarcoplasmic reticulum Ca(2+)-ATPase: do lanthanide ions bind to the calcium transport site?

Gd3+ binding sites on the purified Ca(2+)-ATPase of sarcoplasmic reticulum were characterized at 2 and 6 degrees C and pH 7.0 under conditions in which 45Ca2+ and 54Mn2+ specifically labeled the calcium transport site and the catalytic site of the enzyme, respectively. We detected several classes of Gd3+ binding sites that affected enzyme function: (a) Gd3+ exchanged with 54Mn2+ of the 54MnATP complex bound at the catalytic site. This permitted slow phosphorylation of the enzyme when two Ca2+ ions were bound at the transport site. The Gd3+ ion bound at the catalytic site inhibited decomposition of the ADP-sensitive phosphoenzyme. (b) High-affinity binding of Gd3+ to site(s) distinct from both the transport site and the catalytic site inhibited the decomposition of the ADP-sensitive phosphoenzyme. (c) Gd3+ enhanced 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence in NBD-modified enzyme by probably binding to the Mg2+ site that is distinct from both the transport site and the catalytic site. (d) Gd3+ inhibited high-affinity binding of 45Ca2+ to the transport site not by directly competing with Ca2+ for the transport site but by occupying site(s) other than the transport site. This conclusion was based mainly on the result of kinetic analysis of displacement of the enzyme-bound 45Ca2+ ions by Gd3+ and vice versa, and the inability of Gd3+ to phosphorylate the enzyme under conditions in which GdATP served as a substrate. These results strongly suggest that Ln3+ ions cannot be used as probes to structurally and functionally characterize the calcium transport site on the Ca(2+)-ATPase.     

Conformational studies of Escherichia coli ribosomes with the use of acridine orange as a probe

The interaction of E. coli vacant ribosomes with acridine orange (AO) was studied, to obtain conformational information about rRNAs in ribosomes. Acridine orange binds to an RNA in two different modes: cooperative outside binding with stacking of bound AO's and intercalation between nucleotide bases. Free 16S and 23S rRNAs have almost identical affinities to AO. At 1 mM Mg2+, AO can achieve stacking binding on about 40% of rRNA phosphate groups. The number of stacking binding sites falls to about 1/3 in the 30S subunit in comparison with free 16S rRNA. In the 50S subunit, the number of stacking binding sites is only 1/5 in comparison with free 23S rRNA. Mg2+ ions are more inhibitory for the binding of AO to ribosomes than to free rRNAs. The strength of stacking binding appears to be more markedly reduced by Mg2+ in active ribosomes than in rRNAs. "Tight couple" 70S particles are less accessible for stacking binding than free subunits. The 30S subunits that have irreversibly lost the capability for 70S formation under low Mg2+ conditions have an affinity to AO that is very different from that of active 30S but similar to that of free rRNA, though the number of stacking binding sites is little changed by the inactivation. 70S and 30S ribosomes with stacking bound AO's have normal sedimentation constants, but the 50S subunits reversibly form aggregates.     

Two pathways for phosphate/water oxygen exchange by yeast inorganic pyrophosphatase

A scheme of Mg2+ and Pi binding to yeast inorganic pyrophosphatase has been deduced from the concentration dependencies of the rate of oxygen exchange between Pi and water. The exchange reaction requires the binding of MgPi and free Pi (pathway I) or two MgPi (pathway II) in addition to two Mg2+ ions bound in the absence of Pi. Pathway II predominates above 0.16 mM Mg2+. The rate of formation of bound PPi from bound Pi for pathway II is three times as high as that for pathway I. The results suggest that the binding of the fourth Mg2+ ion to pyrophosphatase stimulates its synthetic vs its hydrolytic capability.     

Biochemical detection of monovalent metal ion binding sites within RNA

Many RNAs, including the ribosome, RNase P, and the group II intron, explicitly require monovalent cations for activity in vitro. Although the necessity of monovalent cations for RNA function has been known for more than a quarter of a century, the characterization of specific monovalent metal sites within large RNAs has been elusive. Here we describe a biochemical approach to identify functionally important monovalent cations in nucleic acids. This method uses thallium (Tl+), a soft Lewis acid heavy metal cation with chemical properties similar to those of the physiological alkaline earth metal potassium (K+). Nucleotide analog interference mapping (NAIM) with the sulfur-substituted nucleotide 6-thioguanosine in combination with selective metal rescue of the interference with Tl+ provides a distinct biochemical signature for monovalent metal ion binding. This approach has identified a K+ binding site within the P4-P6 domain of the Tetrahymena group I intron that is also present within the X-ray crystal structure. The technique also predicted a similar binding site within the Azoarcus group I intron where the structure is not known. The approach is applicable to any RNA molecule that can be transcribed in vitro and whose function can be assayed.     

Structure and thermodynamics of metal binding in the P5 helix of a group I intron ribozyme.

The solution structure of an RNA hairpin modelling the P5 helix of a group I intron, complexed with Co(NH3)63+, has been determined by nuclear magnetic resonance. Co(NH3)63+, which possesses a geometry very close to Mg(H2O)62+, was used to identify and characterize a Mg2+binding site in the RNA. Strong and positive intermolecular nuclear Overhauser effect (NOE) cross-peaks define a specific complex in which the Co(NH3)63+molecule is in the major groove of tandem G.U base-pairs. The structure of the RNA is characterized by a very low twist angle between the two G.U base-pairs, providing a flat and narrowed major groove. The Co(NH3)63+, although highly localized, is free to rotate to hydrogen bond in several ways to the O4 atoms of the uracil bases and to N7 and O6 of the guanine bases. Negative and small NOE cross-peaks to other protons in the sequence reveal a non-specific or delocalized interaction, characterized by a high mobility of the cobalt ion. Mn2+titrations of P5 show specific broadening of protons of the G.U base-pairs that form the metal ion binding site, in agreement with the NOE data from Co(NH3)63+. Binding constants for the interaction of Co(NH3)63+and of Mg2+to P5 were determined by monitoring imino proton chemical shifts during titration of the RNA with the metal ions. Dissociation constants are on the order of 0.1 mM for Co(NH3)63+and 1 mM for Mg2+. Binding studies were done on mutants with sequences corresponding to the three orientations of tandem G.U base-pairs. The affinities of Co(NH3)63+and Mg2+for the tandem G.U base-pairs depend strongly on their sequences; the differences can be understood in terms of the different structures of the corresponding metal ion-RNA complexes. Substitution of G.C or A.U for G.U pairs also affected the binding, as expected. These structural and thermodynamic results provide systematic new information about major groove metal ion binding in RNA.     

Identification of the magnesium ion binding site in the catalytic center of Escherichia coli primase by iron cleavage

Magnesium is essential for the catalysis reaction of Escherichia coli primase, the enzyme synthesizing primer RNA chains for initiation of DNA replication. To map the Mg(2+) binding site in the catalytic center of primase, we have employed the iron cleavage method in which the native bound Mg(2+) ions were replaced with Fe(2+) ions and the protein was then cleaved in the vicinity of the metal binding site by adding DTT which generated free hydroxyl radicals from the bound iron. Three Fe(2+) cleavages were generated at sites designated I, II, and III. Adding Mg(2+) or Mn(2+) ions to the reaction strongly inhibited Fe(2+) cleavage; however, adding Ca(2+) or Ba(2+) ions had much less effect. Mapping by chemical cleavage and subsequent site-directed mutagensis demonstrated that three acidic residues, Asp345 and Asp347 of a conserved DPD sequence and Asp269 of a conserved EGYMD sequence, were the amino acid residues that chelated Mg(2+) ions in the catalytic center of primase. Cleavage data suggested that binding to D345 is significantly stronger than to D347 and somewhat stronger than to D269.     

The environment of two metal ions surrounding the splice site of a group I intron.

Several divalent metal ions (Ca2+, Sr2+ and Pb2+) do not promote splicing, but instead induce cleavage at a single site in the conserved group I intron core in the absence of the guanosine cofactor at elevated pH, generating products with 5'-OH and 3'-phosphate ends. The reaction is competed by Mg2+, which does not cleave at this position, but hydrolyses the splice sites producing 3'-OH and 5'-phosphate ends. Mn2+ promotes both core cleavage and splice site hydrolysis under identical conditions, suggesting that two different metal atoms are involved, each responsible for one type of cleavage, and with different chemical and geometric requirements. Based on the core cleavage position and on the previously proposed coordination sites for Mg2+, we propose a structural location for two metal ions surrounding the splice site in the Michel-Westhof three-dimensional model of the group I intron core. The proposed location was strengthened by a first mutational analysis which supported the suggested interaction between one of the metal ions and the bulged residue in P7.