UC San Diego

Group II introns are DNA sequences that are interspersed throughout the genomes of organisms from all three domains of life. In many cases these intron genes interrupt coding sequences referred to as exons. In order for the exons to be expressed properly, the intervening intron sequence must first be removed. Once transcribed into pre-mRNA, the scattered introns become catalytic ribozymes and are able to efficiently excise themselves and ligate the flanking exons. These RNA molecules contain an active site capable of binding the catalytic metal ions required to perform two sequential transesterification reactions that cut out the intron sequence and paste the exons together. This mechanism is identical to the one used by the spliceosome to process spliceosomal introns from pre-mRNA in eukaryotes. The mechanistic similarities combined with conserved structural elements supports the hypothesis that group II introns and the spliceosome share a common evolutionary ancestor. In addition to their splicing function, group II introns also act as selfish mobile genetic elements known as retrotransposons. These active retroelements contain an open reading frame for a protein called the maturase. When expressed. this protein acts as a folding chaperone by binding specifically to the intron RNA to promote splicing activity. The maturase is a multifunctional protein containing reverse transcriptase and endonuclease domains allowing the group II intron/maturase complex to invade dsDNA through a target primed reverse transcription mechanism. Sequence homology and mechanistic similarities have evolutionarily linked group II introns to non-LTR retroelements, which make up approximately 34% of the human genome. Some of these retroelements are still active and are the cause of many genetic diseases. The focus of this dissertation is the structural study of group II introns to elucidate the mechanism of splicing and retrotransposition. Using cryo-EM, I have been able to obtain density maps at 5.8 Å resolution for a group II intron while splicing and 4.8 Å resolution for a group II intron actively invading dsDNA. The 4.8 Å map represents the first of its kind for any retroelement.