Project 171377

Group II introns are a type of mobile DNA consisting of a self-splicing RNA and a reverse transcriptase (RT). Group II introns are found across the bacterial kingdom, including in many pathogens. Previously, our laboratory identified and classified the varieties of group II introns in bacteria, and identified a surprising diversity of structural forms and inferred mobility behaviors. The most distinctive intron type is "bacterial class C," as these introns insert directly after transcriptional terminator motifs. We have biochemically determined that during an intron insertion reaction in vitro for the B.h.I1 intron of Bacillus halodurans, the DNA target is recognized as a hairpin; however, the in vivo source for the unwound DNA hairpin is unknown. Here, we propose to further explore the diversity of group II introns, both on the global level and for two cases of specialized introns. 1) We will continue database mining to identify, classify and analyze group II introns across bacteria, and the information will be made public on our web site database (www.fw.ucalgary.ca/group2introns/). 2) We will continue investigating the mobility properties of the B.h.I1 intron in vitro by determining the protein binding site on the intron RNA, and by probing how the DNA hairpin of the target is recognized. Additional in vivo studies will examine how the mobility mechanisms intersect with the cellular processes of transcription, transcriptional termination, and DNA replication. 3) We will characterize a novel group II intron derivative that exhibits alternative splicing in vivo. The intron Cl.t.I1 of Clostridium tetanus produces four alternatively spliced forms of a surface layer protein. We will address whether alternative splicing is regulated and how the intron chooses among the different 3' splice sites. Together, these investigations will advance our understanding of group II introns in bacteria, and help to infer how a selfish DNA has adapted over time.

database mining and biochemical/in vivo studies to investigate the diversity, mobility, and splicing of group II introns in bacteria