A UV-INDUCED, MG2+-DEPENDENT CROSS-LINK TRAPS AN ACTIVE FORM OF DOMAIN-3 OF A SELF-SPLICING GROUP-II INTRON

In vitro irradiation of al5 gamma group II intron RNA with low doses of 254 nm UV light induces a single major crosslink. This crosslink was mapped within the domain 3 substructure of this RNA and one of the participating nucleotides was identified. When an RNA containing only the domain 3 substructure is irradiated under the same conditions, an intramolecular crosslink forms between two specific pyrimidines, one of them identical to the nucleotide crosslinked in the full-length intron RNA. In both RNAs, the crosslink is magnesium ion-dependent and photoreversible. A trans assay for domain 3 function was developed and used to find that the crosslinked domain 3 RNA remains highly reactive. This suggests that crosslinking has trapped a functional, Mg2+-induced folded state of this group II intron substructure and that this folding is probably independent of the other domains of the intron.