Hydrolytic cleavage by a group I intron ribozyme is dependent on RNA structures not important for splicing.
DiGIR2 is the group I splicing-ribozyme of the mobile twin-ribozyme intron Dir.S956-1, present in Didymium nuclear ribosomal DNA. DiGIR2 is responsible for intron excision, exon ligation, 3'-splice site hydrolysis, and full-length intron RNA circle formation. We recently reported that DiGIR2 splicing (intron excision and exon ligation) competes with hydrolysis and subsequent full-length intron circularization. Here we present experimental evidence that hydrolysis at the 3'-splice site in DiGIR2 is dependent on structural elements within the P9 subdomain not involved in splicing. Whereas the GCGA tetra-loop in P9b was found to be important in hydrolytic cleavage, probably due to tertiary RNA-RNA interactions, the P9.2 hairpin structure was found to be essential for hydrolysis. The most important positions in P9.2 include three adenosines in the terminal loop (L9.2) and a consensus kink-turn motif in the proximal stem. We suggest that the L9.2 adenosines and the kink-motif represent key regulatory elements in the splicing and hydrolytic reaction pathways.
Department of Molecular Biotechnology, Institute of Medical Biology, University of Tromsø, Norway.
Peik
P
Haugen
Morten
M
Andreassen
Asa B
AB
Birgisdottir
Steinar
S
Johansen
D006868:Hydrolysis; D007438:Introns; D009690:Nucleic Acid Conformation; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000378:metabolism*; D012331:RNA, Fungal / Q000378:metabolism*; D014158:Transcription, Genetic
D016337:RNA, Catalytic; D012331:RNA, Fungal