MORE THAN ONE-WAY TO SPLICE AN RNA - BRANCHING WITHOUT A BULGE AND SPLICING WITHOUT BRANCHING IN GROUP-II INTRONS

Domain 6 (D6) of group II introns contains a bulged adenosine that ser ves as the branch-site during self-splicing. In addition to this adeno sine, other structural features in D6 are likely to contribute to the efficiency of branching. To understand their role in promoting self-sp licing, the branch-site and surrounding nucleotides were mutagenized. Detailed kinetic analysis on the self-splicing efficiency of the mutan ts revealed several interesting features. First, elimination of the br anch-site does not preclude efficient splicing, which takes place inst ead through a hydrolytic first step. Second, pairing of the branch-sit e does not eliminate branching, particularly if the adenosine is invol ved in a mispair. Third, the G-U pairs that often surround group II in tron branch-points contribute to the efficiency of branching. These re sults suggest that there is a strong driving force for promoting self- splicing by group II introns, which employ a versatile set of differen t mechanisms for ensuring that splicing is successful. In addition, th e behavior of these mutants indicates that a bulged adenosine per se i s not the important determinant for branch-site recognition in group I I introns. Rather, the data suggest that the branch-site adenosine is recognized as a flipped base, a conformation that can be promoted by a variety of different substructures in RNA and DNA.