EcI5, a group IIB intron with high retrohoming frequency: DNA target site recognition and use in gene targeting.
We find that group II intron EcI5, a subclass CL/IIB1 intron from an Escherichia coli virulence plasmid, is highly active in retrohoming in E. coli. Both full-length EcI5 and an EcI5-DeltaORF intron with the intron-encoded protein expressed separately from the same donor plasmid retrohome into a recipient plasmid target site at substantially higher frequencies than do similarly configured Lactococcus lactis Ll.LtrB introns. A comprehensive view of DNA target site recognition by EcI5 was obtained from selection experiments with donor and recipient plasmid libraries in which different recognition elements were randomized. These experiments suggest that EcI5, like other mobile group II introns, recognizes DNA target sequences by using both the intron-encoded protein and base-pairing of the intron RNA, with the latter involving EBS1, EBS2, and EBS3 sequences characteristic of class IIB introns. The intron-encoded protein appears to recognize a small number of bases flanking those recognized by the intron RNA, but their identity is different than in previously characterized group II introns. A computer algorithm based on the empirically determined DNA recognition rules enabled retargeting of EcI5 to integrate specifically at 10 different sites in the chromosomal lacZ gene at frequencies up to 98% without selection. Our findings provide insight into modes of DNA target site recognition used by mobile group II introns. More generally, they show how the diversity of mobile group II introns can be exploited to provide a large variety of different target specificities and potentially other useful properties for gene targeting.
Zhuang
Fanglei
F
Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.
Karberg
Michael
M
Perutka
Jiri
J
Lambowitz
Alan M
AM
D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting; D020071:Interspersed Repetitive Sequences; D007438:Introns; D010957:Plasmids / Q000235:genetics