R. Saldanha et al., RNA and protein catalysis in group II intron splicing and mobility reactions using purified components, BIOCHEM, 38(28), 1999, pp. 9069-9083
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.