Large Ribozymes

Key characteristics of large ribozymes:

• Metal ion dependence: Catalytic activity typically requires divalent metal ions, primarily Mg²⁺, which stabilize the ribozyme structure and participate in catalysis.
• Reaction mechanism: These ribozymes catalyze reactions that generate products with 3' hydroxyl and 5' phosphate groups, essential for RNA processing.
• Complex structural organization: Extensive tertiary interactions give rise to highly structured domain architectures, essential for their function.

Group II Introns: evolutionary ancestors of the spliceosome

Group II introns, which range in length from 400 to 1000 nucleotides, are thought to be the evolutionary ancestors of the spliceosome, the cellular machinery responsible for pre-mRNA splicing. These self-splicing ribozymes excise themselves from precursor mRNAs, leaving behind properly processed transcripts. In addition to their biological significance, group II introns have great potential for applications in biotechnology and gene therapy, such as site-specific genome engineering. A well-characterized example is the Sc.ai5γ intron from Saccharomyces cerevisiae, which adopts a defined three-dimensional structure and efficiently catalyzes its own excision. Our research aims to understand the intricate mechanisms of both splicing and folding in these ribozymes.

To achieve this, we employ:

• Fluorescence-based approaches such as single-molecule Förster resonance energy transfer (smFRET) to track RNA folding in real time and assess catalytic activity.
• Structural techniques including NMR spectroscopy, X-ray crystallography, and molecular dynamics (MD) simulations to gain deeper insights into key structural elements governing function.

By combining biochemical, biophysical, and computational methods, we aim to elucidate the fundamental principles that govern ribozyme activity, ultimately contributing to a better understanding of RNA catalysis and its evolutionary implications.