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

Area of Science:

• Molecular Biology
• Biochemistry
• Mycology

Background:

• Group I introns are self-splicing RNA elements found in various organisms, including pathogenic fungi.
• Understanding the mechanisms of group I intron splicing is crucial for developing targeted antimicrobial strategies.

Purpose of the Study:

• To characterize the substrate recognition mechanism of a group I intron from Candida albicans.
• To investigate the role of tertiary interactions in the binding of the 5' exon substrate.
• To provide a basis for designing oligonucleotide-based therapeutics targeting fungal group I introns.

Main Methods:

• Cloning of group I intron precursor and ribozyme from Candida albicans large subunit rRNA.
• In vitro functional assays to confirm precursor and ribozyme activity.
• Oligonucleotide binding assays to determine dissociation constants.
• Site-directed mutagenesis to analyze the contribution of specific base pairs (G.A, G.U) to binding and tertiary interactions.

Main Results:

• The cloned Candida albicans group I intron precursor and ribozyme are functional.
• A model for 5' exon substrate recognition by the intron was proposed, highlighting tertiary contacts.
• Specific tertiary interactions, including 2'-hydroxyl groups and G.A/G.U base pairs, were identified as critical for substrate binding.
• Mutating the G.A pair to G.C significantly weakened interactions, while mutation to G.U enhanced binding 10-fold.

Conclusions:

• Tertiary interactions play a significant role in the precise alignment and binding of the 5' exon substrate to the group I intron.
• The G.A base pair at the splice junction is important for maintaining optimal tertiary interactions.
• These findings provide a foundation for designing novel oligonucleotide therapeutics that exploit these interactions for enhanced binding and inhibition of fungal group I introns.