Molecular recognition by the Candida albicans group I intron: tertiary interactions with an imino G.A pair facilitate binding of the 5' exon and lower the KM for guanosine.
A G.A pair at position -5 in the P1 helix of the Candida albicans ribozyme contributes to tertiary binding of the 5' exon substrate [Disney, M. D., Haidaris, C. G., and Turner, D. H. (2001) Biochemistry 40, 6507-6519]. Here, the G in the G.A pair is replaced with inosine (I) in both semisynthetic ribozymes and oligonucleotide mimics of the internal guide sequence. Comparisons of oligonucleotide binding affinity for these and other sequences indicate that the G.A pair is in an imino conformation where the exocyclic amine of G contributes approximately 1.4 kcal/mol to tertiary interactions that help dock the ribozyme's P1 helix. Furthermore, replacement of the G.A pair with a G-C pair produces less favorable interactions with the 2'-hydroxyl group at the -3 position and a less favorable K(M) for pG in a ribozyme-catalyzed transesterification reaction. These results are also consistent with the G.A pair promoting docking of the P1 helix into the catalytic core. Evidently, tertiary interactions with the exocyclic amino group of a G in a single G.A pair can increase the equilibrium constant for tertiary folding of RNA by roughly 10-fold at 37 degrees C. Results with a G.U or G.G pair replacing the G.A pair at the -5 position suggest similar tertiary interactions with these pairs.
Disney
Matthew D
MD
Department of Chemistry, Center for Human Genetics and Molecular Pediatric Disease, University of Rochester, Rochester, NY 14627-0216, USA.
Turner
Douglas H
DH
D000225:Adenine / Q000378:metabolism; D001665:Binding Sites; D002176:Candida albicans / Q000201:enzymology*; D005091:Exons; D006147:Guanine / Q000378:metabolism; D006151:Guanosine / Q000378:metabolism*; D007288:Inosine / Q000378:metabolism; D007438:Introns; D007700:Kinetics; D008958:Models, Molecular; D018716:Molecular Mimicry; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000378:metabolism*; D013816:Thermodynamics
C116167:GIR1 ribozyme; D016337:RNA, Catalytic; D006151:Guanosine; D007288:Inosine; D006147:Guanine; D000225:Adenine