Mapping divalent metal ion binding sites in a group II intron by Mn2+- andZn2+-induced site-specific RNA cleavage

The function of group II introns depends on positively charged divalent met al ions that stabilize the ribozyme structure and may be directly involved in catalysis. We investigated Mn2+- and Zn2+-induced site-specific RNA clea vage to identify metal ions that fit into binding pockets within the struct urally conserved bI1 group II intron domains (DI-DVI), which might fulfill essential roles in intron function. Ten cleavage sites were identified in D I, 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 scis sions mediated by Mn2+/Zn2+ are competed for by Mg2+, indicating the existe nce 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 a nd functional assays, comparing wild-type precursor and multiple splicing-d eficient mutants. We identified six regions with binding pockets for ions p resumably playing an important role in bI1 structure stabilization. Remarka bly, assays with DI deletions and branch point mutants revealed the existen ce of one Mg2+ binding pocket near the branching A, which is involved in fi rst-step catalysis. This pocket formation depends on precise interaction be tween the branching nucleotide and the 5' splice site, but does not require exon-binding site 1/intron binding site I interaction. This Me2+ ion might support the correct placing of the branching A into the 'first-step active site'.