INTRON DYNAMICS IN BACTERIA

Wadsworth Center, Menands NY

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Abstract

Group I and group II introns are dynamic genetic elements that splice by different self-catalyzed RNA- based mechanisms. Many are also capable of insertion into DNA through distinctive mobility pathways. For each class of intron, mobility characteristically takes the form of homing, whereby the intron transfers to an intronless DNA allele via a break created by an intron-encoded endonuclease. For group I intron homing, recombination events are strictly DNA-based, whereas group II intron homing, also termed retrohoming, involves RNA at levels of both the template and the cleavage enzyme for mobility. The overall goal of this work is to study in bacteria the DNA-based and RNA-based intron rearrangements of these phylogenetically diverse elements. During the past funding period we made progress in understanding the role of DNA exonucleases in group I intron mobility, and in demonstrating illegitimate double-strand-break repair in intron acquisition, suggesting that such a mechanism might account for intron invasion of ectopic sites. In independent studies, we defined the domain structure and function of StpA, an RNA chaperone that promotes splicing of group I introns. We also determined the mode of action of DsrA, an E. coli regulatory RNA. Finally, in major breakthroughs for the field, we established a group II intron as the first functional retrotransposon in prokaryotes, demonstrating both RNA-based retrohoming and retrotransposition to ectopic sites. In addition to the innate mechanistic importance of these results, the invasiveness of group II introns and their similarities to human retrotransposons and spliceosomal introns have great evolutionary and health significance. For the next funding period we propose to demonstrate transposition of group I introns. Additionally, we will extend our analysis of the RNA chaperone function of StpA and further define its structure and global regulatory activity. We will also build on our mechanistic studies and define accessory molecules required for group II intron retrohoming and retrotransposition. Finally, we will ask evolutionary questions relating to group II intron dispersal, the relationship of group II introns to spliceosomal introns of eukaryotes, and the possible role of group II introns in effecting horizontal gene transfer. Thus, by combining the approaches of genetics, biochemistry and structural analysis, we propose to advance our understanding of intron-related nucleic acid dynamics and intron evolution. [unreadable] .