Solution structure of a metal-binding site in the major groove of RNA complexed with cobalt (III) hexammine (original) (raw)
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A crystallographic study of the binding of 13 metal ions to two related RNA duplexes
Nucleic Acids Research, 2003
Metal ions, and magnesium in particular, are known to be involved in RNA folding by stabilizing secondary and tertiary structures, and, as cofactors, in RNA enzymatic activity. We have conducted a systematic crystallographic analysis of cation binding to the duplex form of the HIV-1 RNA dimerization initiation site for the subtype-A and -B natural sequences. Eleven ions (K + , Pb 2+ , Mn 2+ , Ba 2+ , Ca 2+ , Cd 2+ , Sr 2+ , Zn 2+ , Co 2+ , Au 3+ and Pt 4+ ) and two hexammines [Co (NH 3 ) 6 ] 3+ and [Ru (NH 3 ) 6 ] 3+ were found to bind to the DIS duplex structure. Although the two sequences are very similar, strong differences were found in their cation binding properties. Divalent cations bind almost exclusively, as Mg 2+ , at Hoogsteen' sites of guanine residues, with a cation-dependent af®nity for each site. Notably, a given cation can have very different af®nities for a priori equivalent sites within the same molecule. Surprisingly, none of the two hexammines used were able to ef®ciently replace hexahydrated magnesium. Instead, [Co (NH 3 ) 4 ] 3+ was seen bound by inner-sphere coordination to the RNA. This raises some questions about the practical use of [Co (NH 3 ) 6 ] 3+ as a [Mg (H 2 O) 6 ] 2+ mimetic. Also very unexpected was the binding of the small Au 3+ cation exactly between the Watson±Crick sites of a G-C base pair after an obligatory deprotonation of N1 of the guanine base. This extensive study of metal ion binding using X-ray crystallography signi®cantly enriches our knowledge on the binding of middleweight or heavy metal ions to RNA, particularly compared with magnesium.
Biochemical Detection of Monovalent Metal Ion Binding Sites within RNA
Methods, 2001
many RNAs categorically require monovalent ca-Many RNAs, including the ribosome, RNase P, and the group II intron, tions such as K + and ammonium (NH 4 +) to catalyze explicitly require monovalent cations for activity in vitro. Although the their respective chemical reactions in vitro (8-12). necessity of monovalent cations for RNA function has been known for For example, the ribosome, the group II intron, and more than a quarter of a century, the characterization of specific monovalent metal sites within large RNAs has been elusive. Here we RNase P all require between 200 mM and 2 M K + describe a biochemical approach to identify functionally important for catalytic activity. Monovalent cations are known monovalent cations in nucleic acids. This method uses thallium (Tl +), to provide some of the nonspecific charge neutralizaa soft Lewis acid heavy metal cation with chemical properties similar tion required for the dense packing of RNA domains to those of the physiological alkaline earth metal potassium (K +). Nuclein which anionic backbones are in close proximity otide analog interference mapping (NAIM) with the sulfur-substituted (8). The fact that divalent metal ions cannot compennucleotide 6-thioguanosine in combination with selective metal rescue sate for the absence of monovalent cations suggests of the interference with Tl + provides a distinct biochemical signature that there are specific monovalent cation binding for monovalent metal ion binding. This approach has identified a K + binding site within the P4-P6 domain of the Tetrahymena group I intron sites within RNAs. that is also present within the X-ray crystal structure. The technique also Recent high-resolution crystal structures of RNAs predicted a similar binding site within the Azoarcus group I intron where and RNA-protein complexes have demonstrated the the structure is not known. The approach is applicable to any RNA integral association between RNA and specifically molecule that can be transcribed in vitro and whose function can be coordinated monovalent cation cofactors. Specific assayed. ᭧ 2001 Academic Press binding sites have been identified within the tetraloop receptor of the Tetrahymena group I intron P4-P6 domain (Fig. 1), the 4.5S RNA of the signal recognition particle, a ribosome frameshifting pseu
RNA, 2007
Metal ions play a key role in RNA folding and activity. Elucidating the rules that govern the binding of metal ions is therefore an essential step for better understanding the RNA functions. High-resolution data are a prerequisite for a detailed structural analysis of ion binding on RNA and, in particular, the observation of monovalent cations. Here, the high-resolution crystal structures of the tridecamer duplex r(GCGUUUGAAACGC) crystallized under different conditions provides new structural insights on ion binding on GAAA/UUU sequences that exhibit both unusual structural and functional properties in RNA. The present study extends the repertory of RNA ion binding sites in showing that the two first bases of UUU triplets constitute a specific site for sodium ions. A striking asymmetric pattern of metal ion binding in the two equivalent halves of the palindromic sequence demonstrates that sequence and its environment act together to bind metal ions. A highly ionophilic half that binds six metal ions allows, for the first time, the observation of a disodium cluster in RNA. The comparison of the equivalent halves of the duplex provides experimental evidences that ion binding correlates with structural alterations and groove contraction.
Solution structure and thermodynamics of a divalent metal ion binding site in an RNA pseudokno1
Journal of Molecular Biology, 1999
Identification and characterization of a metal ion binding site in an RNA pseudoknot was accomplished using cobalt (III) hexammine, Co(NH3)63+, as a probe for magnesium (II) hexahydrate, Mg(H2O)62+, in nuclear magnetic resonance (NMR) structural studies. The pseudoknot causes efficient −1 ribosomal frameshifting in mouse mammary tumor virus. Divalent metal ions, such as Mg2+, are critical for RNA structure and function; Mg2+ preferentially stabilizes the pseudoknot relative to its constituent hairpins. The use of Co(NH3)63+ as a substitute for Mg2+ was investigated by ultraviolet absorbance melting curves, NMR titrations of the imino protons, and analysis of NMR spectra in the presence of Mg2+ or Co (NH3)63+. The structure of the pseudoknot-Co(NH3)63+ complex reveals an ion-binding pocket formed by a short, two-nucleotide loop and the major groove of a stem. Co(NH3)63+ stabilizes the sharp loop-to-stem turn and reduces the electrostatic repulsion of the phosphates in three proximal strands. Hydrogen bonds are identified between the Co(NH3)63+ protons and non-bridging phosphate oxygen atoms, 2′ hydroxyl groups, and nitrogen and oxygen acceptors on the bases. The binding site is significantly different from that previously characterized in the major groove surface of tandem G·U base-pairs, but is similar to those observed in crystal structures of a fragment of the 5 S rRNA and the P5c helix of the Tetrahymena thermophila group I intron. Changes in chemical shifts occurred at the same pseudoknot protons on addition of Mg2+ as on addition of Co(NH3)63+, indicating that both ions bind at the same site. Ion binding dissociation constants of approximately 0.6 mM and 5 mM (in 200 mM Na+ and a temperature of 15°C) were obtained for Co(NH3)63+ and Mg2+, respectively, from the change in chemical shift as a function of metal ion concentration. An extensive array of non-sequence-specific hydrogen bond acceptors coupled with conserved structural elements within the binding pocket suggest a general mode of divalent metal ion stabilization of this type of frameshifter pseudoknot. These results provide new thermodynamic and structural insights into the role divalent metal ions play in stabilizing RNA tertiary structural motifs such as pseudoknots.
Cation-Specific Structural Accommodation within a Catalytic RNA
Biochemistry, 2006
Metal ions facilitate the folding of the hairpin ribozyme, but do not participate directly in catalysis. The metal complex cobalt (III) hexaammine supports folding and activity of the ribozyme and also mediates specific internucleotide photocrosslinks, several of which retain catalytic ability. These crosslinks imply that the active core structure organized by [Co(NH 3) 6 ] 3+ is different from that organized by Mg 2+ and that revealed in the crystal structure (1). Residues U+2 and C+3 of the substrate, in particular, adopt different conformations in [Co(NH 3) 6 ] 3+. U+2 is bulged out of loop A and stacked on residue G36, whereas the nucleotide at position +3 is stacked on G8, a nucleobase crucial for catalysis. Cleavage kinetics performed with +2 variants and a C+3 U variant correlate with the crosslinking observations. Variants that decreased cleavage rates in magnesium up to 70fold showed only subtle decreases or even increases in observed rates when assayed in [Co (NH 3) 6 ] 3+. Here, we propose a model of the [Co(NH 3) 6 ] 3+-mediated catalytic core generated by MC-SYM that is consistent with these data. Interactions between cations and RNA molecules are critical for the biological activity of RNA, in that metal ions promote RNA folding events and RNA-catalyzed reactions, including RNA processing reactions and peptide bond formation (2). In the hairpin and hammerhead ribozymes, cations function to facilitate folding into the active conformations, but play little or no direct role in catalysis (3-6). Folding and cleavage activity of the hairpin ribozyme can be supported by high concentrations (>1 M) of monovalent ions (4), moderate concentrations (2 to 20 mM) of magnesium and some other divalent ions (7), or by low concentrations (~1 mM) of the trivalent complex [Co(NH 3) 6 ] 3+.. This complex serves as an analogue of hexahydrated magnesium, in that it cannot make inner-sphere binding interactions with RNA (3). Catalysis by the hairpin ribozyme is preceded by a major conformational change, in which the two domains of the ribozyme-substrate complex come into close association with one another. This docking step is accompanied by changes in the orientation of the Watson-Crick helical elements within the complex, which can be monitored by biochemical and biophysical methods, including FRET, electrophoretic mobility, transient electric birefringence, and hydroxyl radical footprinting (8-10). Concomitantly, extensive interactions between the two major non-helical regions are formed, and result in the positioning of the likely catalytic bases, G8 and A38, at the scissile phosphodiester linkage. These latter conformational changes can be monitored by the photocrosslinking and fluorescence behavior of the affected nucleobases (11). The scope of overall conformational change can be visualized by comparing the NMR
Proceedings of the National Academy of Sciences, 2010
Functionally critical metals interact with RNA through complex coordination schemes that are currently difficult to visualize at the atomic level under solution conditions. Here, we report a new approach that combines NMR and XAS to resolve and characterize metal binding in the most highly conserved P4 helix of ribonuclease P (RNase P), the ribonucleoprotein that catalyzes the divalent metal ion-dependent maturation of the 5′ end of precursor tRNA. Extended X-ray absorption fine structure (EXAFS) spectroscopy reveals that the Zn 2þ bound to a P4 helix mimic is sixcoordinate, with an average Zn-O/N bond distance of 2.08 Å. The EXAFS data also show intense outer-shell scattering indicating that the zinc ion has inner-shell interactions with one or more RNA ligands. NMR Mn 2þ paramagnetic line broadening experiments reveal strong metal localization at residues corresponding to G378 and G379 in B. subtilis RNase P. A new "metal cocktail" chemical shift perturbation strategy involving titrations with CoðNH 3 Þ 3þ 6 , Zn 2þ , and CoðNH 3 Þ 3þ 6 ∕Zn 2þ confirm an inner-sphere metal interaction with residues G378 and G379. These studies present a unique picture of how metals coordinate to the putative RNase P active site in solution, and shed light on the environment of an essential metal ion in RNase P. Our experimental approach presents a general method for identifying and characterizing inner-sphere metal ion binding sites in RNA in solution.
Dynamics and Metal Ion Binding in the U6 RNA Intramolecular Stem–Loop as Analyzed by NMR
Journal of Molecular Biology, 2005
The U6 RNA intramolecular stem-loop (ISL) is a conserved component of the spliceosome, and contains an essential metal ion binding site centered between a protonated adenine, A79, and U80. Correlated with protonation of A79, U80 undergoes a base-flipping conformational change accompanied by significant helical movement. We have investigated the dynamics of the U6 ISL by analyzing the power dependence of 13 C NMR relaxation rates in the rotating frame. The data provide evidence that the conformational transition is centered around an exchange lifetime of 84 ms. The U80 nucleotide displays low internal mobility on the picosecond timescale at pH 7.0 but high internal mobility at pH 6.0, in agreement with the global transition resulting in the base of U80 adopting a looped-out conformation with increased dynamic disorder. A kinetic analysis suggests that the conformational change, rather than adenine protonation, is the rate-limiting step in the pathway of the conformational transition. Two nucleotides, U70 and U80, were found from chemical shift perturbation mapping to interact with the magnesium ion, with apparent K d values in the micromolar to millimolar range. These nucleotides also displayed metal ion-induced elevation of R 1 rates, which can be explained by a model that assumes dynamic metal ion coordination concomitant with an induced higher shielding anisotropy for the base 13 C nuclei. Addition of Mg 2C shifts the conformational equilibrium toward the high-pH (base-stacked) structure, accompanied by a significant drop in the apparent pK a of A79.
International journal of molecular sciences, 2016
Due to the polyanionic nature of RNA, the principles of charge neutralization and electrostatic condensation require that cations help to overcome the repulsive forces in order for RNA to adopt a three-dimensional structure. A precise structural knowledge of RNA-metal ion interactions is crucial to understand the mechanism of metal ions in the catalytic or regulatory activity of RNA. We solved the crystal structure of an octameric RNA duplex in the presence of the di- and trivalent metal ions Ca(2+), Mn(2+), Co(2+), Cu(2+), Sr(2+), and Tb(3+). The detailed investigation reveals a unique innersphere interaction to uracil and extends the knowledge of the influence of metal ions for conformational changes in RNA structure. Furthermore, we could demonstrate that an accurate localization of the metal ions in the X-ray structures require the consideration of several crystallographic and geometrical parameters as well as the anomalous difference map.
Functional Identification of Catalytic Metal Ion Binding Sites within RNA
PLoS Biology, 2005
The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (M A , M B , and M C ) that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-S P phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for M C . Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis. Citation: Hougland JL, Kravchuk AV, Herschlag D, Piccirilli JA (2005) Functional identification of catalytic metal ion binding sites within RNA. PLoS Biol 3(9): e277. (JAP)