A homing DNA endonuclease involved in group I intron splicing

Group I introns are large RNAs that can self-splice in vitro but usually require proteins to function in vivo (Lambowitz and Perlman, 1990). Among the proteins that are known to assist group I intron splicing, maturases are the least characterized. Only a handful of maturases have been identified to date (Lambowitz et al., 1999). Prior to my thesis work, a protein encoded by the intron within the mitochondrial apocytochrome b gene (A.n. COB) of Aspergillus nidulans (I-AniI) was demonstrated to directly assist in splicing its cognate intron in vitro. Furthermore, I-AniI was also shown to be a homing endonuclease that catalyzes site-specific DNA hydrolysis reaction in vitro (Ho et al., 1997). I-AniI remains the only group I intron-encoded protein that provides an opportunity to compare both of these seemingly disparate activities in depth.; The work presented here is a detailed investigation into the mechanism of maturase assisted splicing. First, association and splicing kinetic assays revealed a novel mechanism for protein assisted group I intron splicing where binding of I-AniI induces two conformational changes that occur prior to splicing. Initially, I-AniI binds to the unfolded RNA and forms a labile complex followed by a splicing competent complex. A second study involved exploring the relationship between the DNA endonuclease and RNA maturase activities of I-AniI; competition experiments and mutational studies were performed that indicated I-AniI has a functionally distinct binding site for both of its substrates. Since RNA splicing is likely a secondary adaptation of the protein, these observations support a model in which homing endonucleases may have developed maturase function by utilizing a hitherto "non-functional" protein surface. Finally, I investigated how maturases bind so selectively to their cognate introns. A combination of chemical footprinting and nucleotide analog interference mapping approaches point to putative protein binding sites and illustrate the RNA folding requirements for binding COB pre-RNA. This includes binding to a non-conserved group I intron structure suggesting that the mechanism for maturase assisted splicing may be idiosyncratic, arising multiple times during the course of evolution.