Requirements of a Group I Intron for Reactions at the 3′ Splice Site

Abstract

Structural elements of the Tetrahymena group I intron have been implicated in directing the exon-ligation transesterification reaction (the second step of self-splicing) to the correct 3′ splice site. The 3′ splice site also serves as the reaction site in a distinct transesterification reaction known as G-exchange and in a specific hydrolysis reaction. The dependence of the reactions on the internal guide sequence (IGS), an exon-binding element, was examined. Hydrolysis did not require the IGS, while G-exchange did, suggesting that one of the guanosine-binding sites employed during G-exchange requires the IGS. For the exon ligation reaction, the specification of the 3′ splice site is thought to be influenced by the 3′ exon-IGS pairing P10 and three structural elements in the intron:the universally conserved guanosine residue preceding the 3′ splice site, the long-range pairing P9.0, and the triple stem-loop region composed of P9, P9.1 and P9.2. A systematic mutational study of the three structural elements was performed in order to clarify the mechanism of the specification of the 3′ splice site for the reactions. For both transesterification reactions, the elements are important, with some superfluity in function. The specific hydrolysis reaction, however, shows even more dependence on the individual elements, especially the conserved guanosine. The results are discussed in terms of models for the mechanism of self-splicing.