RNA and protein catalysis in group II intron splicing and mobility reactions using purified components

Group II introns encode proteins with reverse transcriptase activity. These proteins also promote RNA splicing (maturase activity) and then, with the excised intron, form a site-specific DNA endonuclease that promotes intron mobility by reverse splicing into DNA followed by target DNA-primed reverse transcription. Here, we used an Escherichia coli expression system for the Lactococcus lactis group II intron L1.LtrB to show that the intron-encoded protein (LtrA) alone is sufficient for maturase activity, and that RNP par ticles containing only the LtrA protein and excised intron RNA have site-sp ecific DNA endonuclease and target DNA-primed reverse transcriptase activit y. Detailed analysis of the splicing reaction indicates that LtrA is an int ron-specific splicing factor that binds to unspliced precursor RNA with a K -d of less than or equal to 0.12 pM at 30 degrees C. This binding occurs in a rapid bimolecular reaction, which is followed by a slower step, presumab ly an RNA conformational change, required for splicing to occur. Our result s constitute the first biochemical analysis of protein-dependent splicing o f a group II intron and demonstrate that a single intron-encoded protein ca n interact with the intron RNA to carry out a coordinated series of reactio ns leading to splicing and mobility.