Structure-induced strain determining the internal cyclization site in the yeast cobI5 autocatalytic intron: theory and experimental tests.
The terminal intron of the apocytochrome b gene of yeast is endowed with autocatalytic potential in vitro and has been also shown to be capable of internal circularization at the linkage 236-237 from the 5' extremity. The aim of this work is to identify the secondary interaction responsible for shaping and activating the internal cyclization site (ICS). This is done by simulating the sequential folding of an abnormally large fragment of 237 nucleotides which starts at the 5' cleavage site and contains the internal guide sequence (IGS) starting 220 nucleotides into the intron. The resulting portion of the overall structure features the conserved interaction P1 and is compatible with the complete consensus secondary structure for this intron. The structural motif which emerges from sequential folding and is responsible for shaping the ICS is confirmed by analyzing the localization of structure-induced strain in the RNA backbone. It is shown that after the conserved stem P1 has been dismantled, the highest strain in a phosphodiester linkage is localized internally precisely at the experimentally determined cyclization site. Moreover, it is shown that our dynamical model of folding is compatible with the actual reaction pathways. Thus, internal cyclization becomes feasible only after conserved interaction P1 has been dismantled. This last event, in turn, might take place as a consequence of either of the following events: (i) 5' cleavage caused by nucleophilic attack by the GTP-cosubstrate or (ii) excision of the 3' exon after prior formation of P10. This last event, in turn, requires dismantling helix P1. Moreover, event (ii) is necessary for internal circularization since it habilitates the 3' terminal guanosine as a nucleophilic agent.
Fernández
A
A
Departmento de Matemática, Universidad Nacional del Sur, Bahía Blanca, Argentina.
Lewin
A S
AS
Rabitz
H
H
D001059:Apoproteins / Q000235:genetics*; D003500:Cyclization; D003573:Cytochrome b Group / Q000235:genetics*; D045303:Cytochromes b; D007438:Introns / Q000235:genetics*; D009010:Monte Carlo Method; D015003:Yeasts / Q000235:genetics*; D058727:RNA, Small Untranslated
D001059:Apoproteins; D003573:Cytochrome b Group; D045303:Cytochromes b; D058727:RNA, Small Untranslated