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The fidelity of group II intron self-splicing and retrohoming relies on long-range tertiary interactions between the intron and its flanking exons. By single-molecule FRET, we explore the binding kinetics of the most important, structurally conserved contact, the exon and intron binding site 1 (EBS1/IBS1). A comparison of RNA-RNA and RNA-DNA hybrid contacts identifies transient metal ion binding as a major source of kinetic heterogeneity which typically appears in the form of degenerate FRET states. Molecular dynamics simulations suggest a structural link between heterogeneity and the sugar conformation at the exon-intron binding interface. While Mg2+ ions lock the exon in place and give rise to long dwell times in the exon bound FRET state, sugar puckering alleviates this structural rigidity and likely promotes exon release. The interplay of sugar puckering and metal ion coordination may be an important mechanism to balance binding affinities of RNA and DNA interactions in general. Mobile group II introns function as ribozymes to splice and reinsert themselves into DNA, thereby colonizing new genomic regions. Here the authors use single-molecule FRET and molecular dynamics simulations to reveal a structural link between metal ion induced kinetic heterogeneity and the sugar puckers at the exon-intron binding interface. [ABSTRACT FROM AUTHOR]

This Perspective outlines the coordinating properties of lead(ii), to some extent in comparison with related metal ions like Ca2+, Zn2+ or Cd2+. It is worth noting that the affinity of Pb2+ towards phosphate residues corresponds to that of Cu2+. Furthermore, the binding tendency of Pb2+ towards thiophosphate groups as present in methyl thiophosphate (MeOPS2−) or uridine 5′-O-thiomonophosphate (UMPS2−) is compared with that of the parent ligands, that is, methyl phosphate (CH3OPO32−) and uridine 5′-monophosphate (UMP2−). The replacement of an O by a S atom makes the monoprotonated thiophosphate group considerably more acidic [compared to ROP(O)2−(OH)], but at the same time its affinity for Pb2+ increases tremendously: more than 99% of Pb2+ is S-bound. This is very different if the coordinating properties of uridylyl-(5′→3′)-[5′]-uridylate (pUpU3−) and P-thiouridylyl-(5′→3′)-[5′]-uridylate (pUp(S)U3−) are compared. The phosphate-coordinated Pb2+ forms a 10-membered chelate with one of the two terminal O atoms of the phosphodiester linkage, which reaches a formation degree of about 90% in Pb(pUpU)−. However, in Pb(pUp(S)U)− the formation degree of the chelate is reduced to about half in accordance with the fact that now only one terminal O atom is available in the thiophosphate diester bridge, that is, Pb2+ coordinates to this O showing no affinity for S in ROP(O)(S)−OR′. These observations are ascribed to the properties of the Pb2+ lone pair, which shapes the Pb2+ coordination sphere; its role is discussed further in this Perspective and a caveat is made regarding Pb2+ binding to a thiophosphate diester linkage. [ABSTRACT FROM AUTHOR]

Group II introns are large self-splicing ribozymes that require high amounts of monovalent and divalent metal ions for folding and catalysis under in vitro conditions. Domain 6 of these ribozymes contains a highly conserved adenosine whose 2′-OH acts as a nucleophile during self-cleavage via the branching pathway. We have previously suggested a divalent metal ion that binds to the major groove at the GU wobble pair above the branch-A in a minimal, but active branch domain construct (D6-27) from the yeast mitochondrial intron Sc.ai5γ. Here we characterize metal ion binding to the phosphate oxygens at the branch site. In vitro transcription yielded a D6-27 construct where all R oxygens of the uridine phosphate groups are replaced by sulfur (α-thio-D6-27). We determined its NMR structure, the second RNA-only structure containing thiophosphate groups. [P] resonances were assigned and chemical shift changes monitored upon titration with Cd. In addition, the two uridines flanking the branch-point, U19 and U21 were specifically thioated by chemical synthesis (thio-U19-D6-27 and thio-U19/U21-D6-27), enabling us to study Cd binding at the R -, as well as the S - position of the corresponding phosphate oxygens. Our studies reveal that both non-bridging phosphate oxygens of U19 are involved in metal ion coordination, whereas only the major groove phosphate oxygen of U21 is influenced. Together with NOE data of a hexaamminecobalt(III) titration, this suggests a single metal ion binding site at the GU wobble pair above the branch point in the major groove of D6 of this group II intron ribozyme. [ABSTRACT FROM AUTHOR]

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 Ca2+, Mn2+, Co2+, Cu2+, Sr2+, and Tb3+. 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. [ABSTRACT FROM AUTHOR]

Hexaamminecobalt(III), an octahedral, inert metal ion complex, has lately gained increasing attention of structural biologists and bioinorganic chemists due to its use in structure determination of nucleic acids. This complex mimics outer-sphere binding events of the physiologically relevant magnesium(II)hexaaqua ion; hexaamminecobalt(III) often finds usage either in NMR spectroscopy, where cross-peaks between the complex ammines and the nucleic acid protons near the binding site are observed, in X-ray spectroscopy as heavy metal derivative for phasing, or in other techniques. In this review, we discuss the basic hexaamminecobalt(III) binding modes and give an overview on the most recent findings on [Co(NH3)6]Cl3-nucleic acid complexes. The various techniques that are applied in combination with this complex are mentioned and briefly summarized. Special attention is given to the application of [Co(NH3)6]Cl3 in nuclear magnetic resonance spectroscopy of nucleic acids, where it is used to reveal potential outer-sphere magnesium binding sites. [ABSTRACT FROM AUTHOR]

The intrinsic acid-base properties of the hexa-2′-deoxynucleoside pentaphosphate, d(ApGpGpCpCpT) [=(A1⋅G2⋅G3⋅C4⋅C5⋅T6)=(HNPP)5−] have been determined by 1H NMR shift experiments. The p Ka values of the individual sites of the adenosine (A), guanosine (G), cytidine (C), and thymidine (T) residues were measured in water under single-strand conditions (i.e., 10 % D2O, 47 °C, I=0.1 M, NaClO4). These results quantify the release of H+ from the two (N7)H+ (G⋅G), the two (N3)H+ (C⋅C), and the (N1)H+ (A) units, as well as from the two (N1)H (G⋅G) and the (N3)H (T) sites. Based on measurements with 2′-deoxynucleosides at 25 °C and 47 °C, they were transferred to p Ka values valid in water at 25 °C and I=0.1 M. Intramolecular stacks between the nucleobases A1 and G2 as well as most likely also between G2 and G3 are formed. For HNPP three p Ka clusters occur, that is those encompassing the p Ka values of 2.44, 2.97, and 3.71 of G2(N7)H+, G3(N7)H+, and A1(N1)H+, respectively, with overlapping buffer regions. The tautomer populations were estimated, giving for the release of a single proton from five-fold protonated H5(HNPP)±, the tautomers (G2)N7, (G3)N7, and (A1)N1 with formation degrees of about 74, 22, and 4 %, respectively. Tautomer distributions reveal pathways for proton-donating as well as for proton-accepting reactions both being expected to be fast and to occur practically at no 'cost'. The eight p Ka values for H5(HNPP)± are compared with data for nucleosides and nucleotides, revealing that the nucleoside residues are in part affected very differently by their neighbors. In addition, the intrinsic acidity constants for the RNA derivative r(A1⋅G2⋅G3⋅ C4⋅C5⋅U6), where U=uridine, were calculated. Finally, the effect of metal ions on the p Ka values of nucleobase sites is briefly discussed because in this way deprotonation reactions can easily be shifted to the physiological pH range. [ABSTRACT FROM AUTHOR]

Metal-mediated base pairs represent a powerful tool for the site-specific functionalization of nucleic acids with metal ions. The development of applications of the metal-modified nucleic acids will depend on the availability of structural information on these double helices. We present here the NMR solution structure of a self-complementary DNA oligonucleotide with three consecutive imidazole nucleotides in its centre. In the absence of transition-metal ions, a hairpin structure is adopted with the artificial nucleotides forming the loop. In the presence of Ag(i) ions, a duplex comprising three imidazole–Ag+–imidazole base pairs is formed. Direct proof for the formation of metal-mediated base pairs was obtained from 1J(15N,107/109Ag) couplings upon incorporation of 15N-labelled imidazole. The duplex adopts a B-type conformation with only minor deviations in the region of the artificial bases. This work represents the first structural characterization of a metal-modified nucleic acid with a continuous stretch of metal-mediated base pairs. [ABSTRACT FROM AUTHOR]

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 Tb3+ and Mg2+. 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. [ABSTRACT FROM AUTHOR]

The article discusses the findings of the study which examines the metal-ion-binding strength of the hydroxyl group. The study states that the riginity resulting upon the glycinate-type binding brings the hydroxyl group close to the vicinity of the metal ion. It mentions that the combination of a well-suited primary binding site with a large number of hydroxyl group leads to a higher complex stability and increased selectivity.