DNA target site requirements for homing in vivo of a bacterial group II intron encoding a protein lacking the DNA...

Group II intron-encoded proteins (IEPs), which have maturase and reverse transcriptase activities, form a ribonucleoprotein (RNP) complex with the intron RNA. Some IEPs also have a C-terminal DNA-binding region and conserved DNA endonuclease domain involved in the recognition and cleavage of specific DNA target sites used for intron homing. RmInt1 is a mobile group II intron of Sinorhizobium meliloti, the IEP of which lacks the endonuclease domain, as do over half of their bacterial counterparts. Here, we analyzed the DNA target sequence requirements for homing in vivo of intron RmInt1 and compared these requirements to those established for the Lactococcus lactis Ll.LtrB intron, a representative of mobile subgroup IIA introns encoding proteins with functional C-terminal DNA endonuclease domains. As for Ll.LtrB, RmInt1 homing requires modifiable base-pairing interactions between the intron RNA and the DNA target, involving 13 nucleotides. However, instead of the delta-delta' interaction, typical of subgroup IIA introns, we demonstrate that RmInt1 recognizes the first nucleotide within the 3' exon of the target site by a new EBS3/IBS3 pairing predicted for subgroup IIB self-splicing introns. Unlike Ll.LtrB, there are less stringent requirements for RmInt1 recognition of distal 5' and 3' exon regions, where only single nucleotide positions are fixed constraints for intron homing. Our results predict differences in the DNA target-site requirements among group II introns, which may have mechanistic and evolutionary implications.