Determination of the Intrinsic Affinities of Multiple Site-Specific Mg²⁺Ions Coordinated to Domain 6 of a Group II Intron Ribozyme

Abstract

Group II introns are large metallo-ribozymes that use divalent metal ions in folding and catalysis. The 3'-terminal domain 6 contains a conserved adenosine whose 2'-OH acts as the nucleophile in the first splicing step. In the hierarchy of folding, D6 binds last into the active site. In order to investigate and understand the folding process to the catalytically active intron structure, it is important to know the individual binding affinities of Mg2+ ions to D6. We recently studied the solution structure of a 27 nucleotide long domain 6 (D6-27) from the mitochondrial yeast group II intron Sc.ai5, identifying also five Mg2+ binding sites including the one at the 5'-terminal phosphate residues. Mg2+ coordination to the 5'-terminal di-and triphosphate groups is strongest (e.g., log KA,TP = 4.55 ± 0.10) and could be evaluated here in detail for the first time. The other four binding sites within D6-27 are filled simultaneously (e.g., log KA,BR = 2.38 ± 0.06) and thus compete for the free Mg2+ ions in solution having a distinct influence on the individual affinities of the various sites. For the first time we take this competition into account to obtain the intrinsic binding constants, describing a method that is generally applicable. Our data illustrates that any RNA undergoing tertiary contacts to a second RNA needs first to be loaded evenly and specifically with metal ions in order to compensate for the repulsion between the negatively charged RNAs. ions in solution having a distinct influence on the individual affinities of the various sites.For the first time we take this competition into account to obtain the intrinsic binding constants, describing a method that is generally applicable. Our data illustrates that any RNA undergoing tertiary contacts to a second RNA needs first to be loaded evenly and specifically with metal ions in order to compensate for the repulsion between the negatively charged RNAs.3

Effect of the ribose versus 2′-deoxyribose residue on the metal ion-binding properties of purine nucleotides

“…Mg 2+ plays an ubiquitous role in biological systems 1,10 including ribozymes 11,12 having a high affinity towards oxygen donor sites like phosphate groups. [12][13][14] Ni 2+ and Cu 2+ have both a significant affinity towards O and N sites 13,14 and both play important roles in metabolic as well as toxicologic processes. 10,15 We have now measured by potentiometric pH titrations the stability constants of the complexes formed between the mentioned three metal ions and the 2'-deoxyadenosine 5'-phosphates (dNP 2-/3-/4-), whereas those for the corresponding Zn 2+ complexes were estimated.…”

“…Instead, the stabilities of the 'open' species [eqn (11)] can be calculated with the previously determined parameters 29,30 for the straight-line equation (12) and the known acidity constants of the H(NP) -/2-species. However, this is true only for the M 2+ complexes of GMP 2-/dGMP 2-, AMP 2-/dAMP 2-, and ADP 3-/dADP 3-.…”

Metal ion-N7 coordination in a ribozyme branch domain by NMR

“…1). 29,30 However for this site, as well as for the whole group II intron architecture, the exact coordination spheres of Mg 2+ ions remain elusive.…”

Metal Ion–RNA Interactions Studied via Multinuclear NMR

“…Nevertheless, NMR titration experiments of RNA molecules with Mg 2+ allow a detailed examination of simultaneous metal ion binding to various sites, as well as offer the possibility to calculate intrinsic binding affinities (20). In this protocol we first concentrate on the practical aspects of RNA sample preparation for such experiments.…”

“…During transcription, metal ion assisted partial hydrolysis of the terminal NTP seems to occur, leading to a mixture of a 5' terminal tri-and di-phosphate groups. Once the sample is prepared no further change in the ratio of diphosphate and triphosphate terminal groups seems to occur (20).…”