Conjugation as a Highly Sensitive Assay to Study Group II Intron Splicing In Vivo.

Group II introns are noncoding sequences that interrupt genes, and that must be removed or spliced-out at the RNA level during gene expression. Following the transcription of interrupted genes, group II introns self-splice while concurrently ligating their flanking exons to generate mature mRNAs ready for translation. Ll.LtrB, the model group II intron from the gram-positive bacterium Lactococcus lactis, interrupts the gene coding for a relaxase enzyme that initiates the transfer of mobile elements by conjugation. This functional link between group II intron splicing and conjugative transfer enabled us to engineer highly sensitive splicing assays using the native biological context of Ll.LtrB. The splicing efficiency/conjugation assay was developed to determine the splicing competence of various Ll.LtrB mutants, whereas the splicing selection/conjugation assay was established to isolate splicing-proficient variants from a randomly generated bank of mutated introns.