The proposed research involves continued biochemical-genetics studies of the protein-dependent splicing and mobility reactions of group II introns. Group II intron mobility is mediated by intron-encoded reverse transcriptases that also function as maturases to promote splicing of the intron. During the current grant period, we elucidated the general nature of the protein- dependent splicing and mobility reactions, and developed tools for studying the reactions further. First, we showed that group II intron mobility ( homing ) occurs via target-DNA primed reverse transcription mechanisms involving a novel intron-encoded DNA endonuclease, which is an RNP complex containing the intron- encoded protein and the excised intron RNA. In one mechanism, the intron RNA in the RNP complex reverse splices directly into the DNA target site and is then reverse transcribed by the intron-encoded protein. Second, we showed that group II intron endonucleases, which determine the site of intron insertion, use both their RNA and protein subunits to recognize specific sequences in their DNA target sites. In the case of the intron RNA, this involves base pairing between a 13-nt region of the DNA target site and sequences in the intron RNA and consequently, it is possible to change the specificity of the endonuclease simply by modifying the intron RNA. These findings raise the possibility that group II introns might be used as tools for targeted gene disruption and insertion of genetic information at desired sites in DNA genomes. Finally, we developed an efficient E. coli expression system for the Lactococcus lactis group II intron L1.LtrB. Recent extensions of the work include the development of genetic systems for studying both the retrohoming and splicing of the Lactococcal intron in E. coli, and methods for studying the splicing and mobility reactions using purified components.
Specific aims are: (1) To continue biochemical analysis of the maturase-dependent group II intron splicing reaction. (2) To identify additional protein factors involved in group II intron splicing. (3) To analyze the reaction mechanism and DNA target-site interactions of the intron-encoded DNA endonuclease activity. (4) To support collaborations to determine X-ray crystal structures of the group II intron-encoded protein and its complexes with intron RNA and RT substrates. (5) To continue collaborations on group II intron mobility mechanisms in yeast mitochondria and bacteria. (6) To explore the feasibility of using group II introns as vectors for targeted gene disruption and site-specific DNA insertion in animal cells.
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