| Abstract
| - The Tetrahymena group I ribozyme's oligonucleotide substrate, CCCUCUA5, forms six basepairs with the ribozyme's internal guide sequence (IGS, 5‘GGAGGG) to give the P1 duplex, and thisduplex then docks into the active site via tertiary interactions. Shortening the substrate by three residuesto give UCUA5 reduces the equilibrium constant for P1 docking by ∼200-fold even though UCUA5 retainsall the functional groups known to be involved in tertiary interactions [Narlikar, G. J., Bartley, L. E.,Khosla, M., and Herschlag, D. (1999) Biochemistry38, 14192−14204]. Here we show that the P1 duplexformed with UCUA5 engages in all of the major tertiary interactions made by the standard P1 duplex.This suggests that the destabilization is not due to disruption of specific tertiary interactions. It thereforeappears that the weaker docking of UCUA5 arises from the increased conformational freedom of theundocked P1 duplex, which has three unpaired IGS residues and thus a larger entropic cost for docking.Further, a 2‘-methoxy substitution at an IGS residue that is base-paired in the standard P1 duplex withCCCUCUA5 but unpaired in the P1 duplex with UCUA5 destabilizes docking of the standard P1 duplex∼300-fold more than it destabilizes docking of the P1 duplex formed with UCUA5. These results suggestthat fixation of groups in the context of a rigid duplex may be a general strategy used by RNA tosubstantially increase interaction specificity, both by aiding binding of the desired functional groups andby increasing the energetic cost of forming alternative interactions.
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