Binding of guanosine and 3' splice site analogues to a group I ribozyme: interactions with functional groups of guanosine and with additional nucleotides.
Dissociation constants, Kd, were measured by equilibrium dialysis at 5 degrees C for a series of substrates binding to the L-21 ScaI ribozyme derived from the Tetrahymena thermophila self-splicing large subunit (LSU) ribosomal RNA intron. These substrates are analogues for the 3' exon splice site, the cyclization site, and the exogenous G that initiates group I splicing. UCG has a Kd of 17 microM. Lengthening the substrate to GUCG and GGUCG enhances binding but by less than expected from potential base pairing. Functional groups on the 3'-terminal G of GUCG were replaced with H to test their effect on binding. GUC(2'dG) binds slightly tighter than the all-ribose molecule but shows no reactivity as a substrate. GUC(3'dG) binds weaker than GUCG. Inosine and 2-aminopurine ribonucleoside at the 3' position weaken binding by 16- and 26-fold, respectively, but both tetramers are reactive. Thus hydrogen bonds to Watson-Crick pairing positions of the 3'G of GUCG contribute 1-2 kcal/mol to the free energy change for binding. Similar results are found in comparisons of UCG with UC(2'dG), UC(3'dG), and UCI. The nonreactive substrate GUCdGA includes a phosphodiester bond 3' to the guanosine that is the site of chemistry for the all-ribose substrate GUCGA; GUCdGA binds 50 times more weakly than GUCdG. A similar result is obtained for GUCdGU. Competition experiments show that guanosine and guanosine 5'-monophosphate bind with dissociation constants of about 0.9 mM. The monomers 2'dG and 3'dG have Kd's of 0.5 and > or = 3 mM, respectively. This suggests that sugar pucker and/or interactions with hydroxyl groups affect binding. Implications for ribozyme catalysis, splicing, cyclization, and design of antisense oligomers are discussed.
Department of Chemistry, University of Rochester, New York 14627-0216.
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D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D003956:Dialysis; D006151:Guanosine / Q000737:chemistry / Q000378:metabolism*; D006358:Hot Temperature; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000378:metabolism*; D013329:Structure-Activity Relationship; D016808:Tetrahymena thermophila / Q000201:enzymology; D013816:Thermodynamics
D009711:Nucleotides; D016337:RNA, Catalytic; D006151:Guanosine