INTRON DYNAMICS IN THE PHAGE/E COLI SYSTEM

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The phylogenetically widespread group I and group II introns are dynamic gentic elements. They splice by different self-catalyzed RNA- based mechanisms, while many are also capable of insertion into DNA through distinct mobility pathways. For each calss of intron, mobility characteristically takes the form of 'homing', whereby the intron transfers to an intronless DNA allele via a double-strand break created by an intron-encoded endonuclease. Whereas for group I homing, recombination events are strictly DNA-based, group II homing involves RNA at levels of both the template and the cleavage enzyme for mobility. The overall goal of this work is to use prokaryotic genetic systems to study both DNA-based and RNA-based intron rearrangements. During the past funding period we made progress towards defining structural and functional requirements of group I intron homing in the T4 phage system and determined that the process occurs by multiple recombination pathways. Furthermore, we advanced our understanding of protein-assisted RNA-based reactions by identifying E. Coli proteins that exhibit RNA chaperone activity in vitro. We also obtained evidence that supports an gypothesis for how mobile group I introns evolved, by invasion of an endonuclease gene into DNA encoding the self-splicing intron. For the next funding period we propose to extend the studies defining the group I mobility process. We will also continue our work on protein-assisted splicing, by probing the mechanism of StpA, an E. Coli protein with RNA chaperone activity, and by analyzing the Neurospora splicing effector CYT-18 in E. coli. Additionally, we wish to extend our work to studying bacterial group II intron homing, a process dependent upon both RNA splicing and DNA recombination. Thus, by exploiting eubacterial genetic systems and combining the approaches of genetic analysis, biochemical characterization and structural study, we propose to advance our understanding of intron- related nucleic acid dynamics.