INTRON DYNAMICS IN BACTERIA

Wadsworth Center, Menands NY

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Abstract

EXCEED THE SPACE PROVIDED. The proposed research is a continued study of mobile introns in bacteria. The mobile group I and group II introns are both self-splicing, but their RNA-splicing and DNA-mobility pathways are distinct. In each case, the intron typically transfers to an allelic intron-less site, in a homingprocess initiated by intron-encoded endonucleases. For group I intron homing, recombination events are strictly DNA-based whereas for group II intron homing, also termed retrohoming, RNA is involved at levels of both the intron template and the cleavage enzyme for mobility. Transposition to ectopic sites also occurs at low frequency, and is the process responsible for intron dissemination in nature. During the current funding period we showed the following: Homing of the group I td intron is regulated by the very endonuclease that promotes the process, by acting as an autorepressor. Also, retrohoming of group II introns is dependent on host polymerases in a way that suggests that intron movement is related to the cell's DNA damage response. Furthermore, group II intron retrotransposition occurs into single- or double-stranded DNAs in a host-dependent fashion. Additionally, transposed group II introns retain their ability to move, reflecting the high fidelity of the group II intron- encoded reverse transcriptase. Finally, in a study of a small regulatory RNA, DsrA, included in a non-intron- related specific ami, we showed not only that DsrA undergoes conformational changes to effect global regulation, but also that DsrA promotes acid tolerance in pathogenic bacteria, thereby enhancing virulence. In the upcoming funding period we will follow up on these findings using genetic studies in bacteria, as well as biochemical and structural approaches. Further, we will address exciting questions relating to intron evolution and the relationship of group II nitrons to spliceosomeal introns in yeast, all as described in the original application. Our overall goal of studying DNA- and RNA-based rearrangements of model introns remains unchanged. In addition to the innate mechanistic importance of these studies, the invasiveness of group II introns and their similarities to human retrotransposons and spliceosomal introns have great evolutionary, biotechnological and medical significance.