Photocrosslinking of 4-thio uracil-containing RNAs supports a side-by-sidearrangement of domains 5 and 6 of a group II intron

Previous studies suggested that domains 5 and 6 (D5 and D6) of group II int rons act together in splicing and that the two helical structures probably do not interact by helix stacking. Here, we characterized the major Mg2+ io n- and salt-dependent, long-wave UV light-induced, intramolecular crosslink s formed in 4-thiouridine-containing D56 RNA from intron 5 gamma (al5 gamma ) of the COXl gene of yeast mtDNA. Four major crosslinks were mapped and fo und to result from covalent bonds between nucleotides separating D5 from D6 [called 5(56)] and residues of D6 near and including the branch nucleotide . These findings are extended by results of similar experiments using 4-thi oU containing D56 RNAs from a mutant allele of al5 gamma and from the group IIA intron, ail. Trans-splicing experiments show that the crosslinked wild -type al5 gamma D56 RNAs are active for both splicing reactions, including some first-step branching. An RNA containing the 3-nt 5(56) sequence and D6 of al5 gamma yields one main crosslink that is Identical to the most minor of the crosslinks obtained with D56 RNA, but in this case in a cation-inde pendent fashion. We conclude that the interaction between 5(56) and D6 is i nfluenced by charge repulsion between the D5 and D6 helix backbones and tha t high concentrations of cations allow the helices to approach closely unde r self-splicing conditions. The interaction between J(56) and D6 appears to be a significant factor establishing a side-by-side (i.e., not stacked) or ientation of the helices of the two domains.