Determination of the intrinsic affinities of multiple site-specific Mg(2+) ions coordinated to domain 6 of a group II intron ribozyme.
Group II introns are large metallo-ribozymes that use divalent metal ions in folding and catalysis. The 3'-terminal domain 6 (D6) contains a conserved adenosine whose 2'-OH group 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 D6 (D6-27) from the mitochondrial yeast group II intron Sc.ai5gamma, also identifying 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 is 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 molecule undergoing tertiary contacts to a second RNA molecule first needs to be loaded evenly and specifically with metal ions to compensate for the repulsion between the negatively charged RNA molecules.
Erat
Michèle C
MC
Institute of Inorganic Chemistry, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland.
Sigel
Roland K O
RK
D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000737:chemistry; D007438:Introns / Q000235:genetics*; D008274:Magnesium / Q000737:chemistry*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002413:Cations, Divalent; D010710:Phosphates; D016337:RNA, Catalytic; D008274:Magnesium