Recognition elements for 5 ' exon substrate binding to the Candida albicans group I intron

A group I intron precursor and ribozyme were cloned from the large subunit rRNA of the human pathogen Candida albicans. Both the precursor and ribozym e are functional as determined from in vitro assays. Comparisons of dissoci ation constants for oligonucleotide binding to the ribozyme and to a hexanu cleotide mimic of its internal guide sequence lead to a model for recogniti on of the 5' exon substrate by this intron. In particular, tertiary contact s with the P1 helix that help align the splice site include three 2'-hydrox yl groups, a G.U pair that occurs at the intron's splice junction, and a G. A pair. The free energy contribution that each interaction contributes to t ertiary binding is determined. When the G.A pair is replaced with a G-C pai r, tertiary interactions to 5' exon mimic 2'-hydroxyl groups are significan tly weakened. When the G.A pair is replaced with a G.U pair, tertiary inter actions are retained and binding is 10-fold tighter. These results expand o ur knowledge of substrate recognition by group I introns, and also provide a basis for rational design of oligonucleotide-based therapeutics for targe ting group I introns by binding enhancement by tertiary interactions and su icide inhibition strategies.