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'.
Hertweck
M
M
Vienna BioCenter, Institute of Microbiology and Genetics, Austria.
Mueller
M W
MW
D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D007438:Introns / Q000187:drug effects*; D008345:Manganese / Q000494:pharmacology*; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D022821:RNA Splice Sites; D012326:RNA Splicing / Q000187:drug effects*; D020871:RNA Stability; D012441:Saccharomyces cerevisiae / Q000235:genetics; D015032:Zinc / Q000494:pharmacology*
D002413:Cations, Divalent; D022821:RNA Splice Sites; D008345:Manganese; D012313:RNA; D015032:Zinc