Selection of host factors that stimulate splicing of the Ll.LtrB group II intron in Lactococcus lactis.

CECILIA QUIROGA; YOUNG IM; BENOIT COUSINEAU

Congreso; Riboclub Annual Metting; 2010

Group II (GII) introns are large ribozymes that fold into a highly conserved RNA secondary structure of six domains (D1-D6) radiating from a central wheel. The Ll.LtrB GII intron from the gram-positive bacterium Lactococcus lactis harbors an open reading frame (ORF) within D4 which encodes for LtrA, a multifunctional protein with reverse transcriptase, maturase and endonuclease activities. The maturase activity of LtrA is essential to promote proper intron folding and splicing in vivo. Ll.LtrB interrupts the relaxase gene (ltrB) of an integrative and conjugative element, named the sex factor (SF). The relaxase enzyme recognizes the origin of transfer, nicks the DNA and initiates conjugative transfer of the SF. Therefore, conjugative transfer of the SF depends on the efficient splicing of Ll.LtrB from the relaxase pre-mRNA. Using a splicing/conjugation assay we found that in the absence of LtrA, Ll.LtrB splicing is greatly compromised. We then screened a genomic library by conjugation for L. lactis host factors that would complement the LtrA maturase activity and stimulate Ll.LtrB splicing. Out of the 14 library candidates analyzed, 6 were shown to significantly stimulate Ll.LtrB splicing. Our data suggest that some protein factors encoded in the L. lactis genome have maturase activity and can partially restore the splicing activity of the Ll.LtrB GII intron deleted for its intron-encoded splicing co-factor, LtrA.