Guanosine binding to the Tetrahymena ribozyme: thermodynamic coupling with oligonucleotide binding.
The L-21 Sca I ribozyme derived from the group I intron of Tetrahymena thermophila pre-rRNA catalyzes an endonuclease reaction analogous to the first step of self-splicing. Guanosine (G) is bound by the ribozyme, and its 3'-hydroxyl group acts as the nucleophile. Here, we provide evidence that Km for G in several single-turnover reactions is equal to the equilibrium dissociation constant for G. This evidence includes the observation that removal of the 2'-hydroxyl group at the cleavage site of the oligoribonucleotide substrate [from CCCUCUA to CCCUC(dU)A] decreases the rate of cleavage approximately 1000-fold but has no effect on either the Km for G (0.17 mM) or for guanosine 5'-monophosphate (pG) (0.09 mM). In the course of this study, it was observed that Km for G or pG was lower by a factor of 5 for reactions with the ribozyme-CCCUC(dU)A complex compared with the free ribozyme, indicating a modest amount of thermodynamic coupled binding of the two substrates. The decrease in the rate of oligonucleotide dissociation upon addition of saturating pG provides independent support for this coupling. Coupling is lost with a substrate that cannot make the normal tertiary interactions with the ribozyme, providing evidence that coupled binding requires docking of the substrate into the catalytic core. Surprisingly, the binding of product CCCUCU and G is slightly anticooperative, indicating that the cleaved pA is important for coupling with substrate. Coupled binding suggests a splicing model in which the intron binds G tightly to promote the first step of reaction, after which its binding is an order of magnitude weaker, thereby facilitating the second step.
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, 80309-0215.
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D006151:Guanosine / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D013379:Substrate Specificity; D016808:Tetrahymena thermophila / Q000378:metabolism*; D013816:Thermodynamics
D009841:Oligonucleotides; D016337:RNA, Catalytic; D006151:Guanosine