Group II intron endonucleases use both RNA and protein subunits for recognition of specific sequences in double-stranded DNA.
Group II introns use intron-encoded reverse transcriptase, maturase and DNA endonuclease activities for site-specific insertion into DNA. Remarkably, the endonucleases are ribonucleoprotein complexes in which the excised intron RNA cleaves the sense strand of the recipient DNA by reverse splicing, while the intron-encoded protein cleaves the antisense strand. Here, studies with the yeast group II intron aI2 indicate that both the RNA and protein components of the endonuclease contribute to recognition of an approximately 30 bp DNA target site. Our results lead to a model in which the protein component first recognizes specific nucleotides in the most distal 5' exon region of the DNA target site (E2-21 to -11). Binding of the protein then leads to DNA unwinding, enabling the intron RNA to base pair to a 13 nucleotide DNA sequence (E2-12 to E3+1) for reverse splicing. Antisense-strand cleavage requires additional interactions of the protein with the 3' exon DNA (E3+1 to +10). Our results show how enzymes can use RNA and protein subunits cooperatively to recognize specific sequences in double-stranded DNA.
Guo
H
H
Department of Molecular Genetics, The Ohio State University, 484 West Twelfth Avenue, Columbus, OH 43210-1292, USA.
Zimmerly
S
S
Perlman
P S
PS
Lambowitz
A M
AM
D001483:Base Sequence; D004271:DNA, Fungal / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D007438:Introns; D008928:Mitochondria / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism
D004271:DNA, Fungal; D016337:RNA, Catalytic; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; D003576:Electron Transport Complex IV; C066564:aI2 protein, S cerevisiae; D012194:RNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases; D015032:Zinc