Use of duplex rigidity for stability and specificity in RNA tertiary structure

The Tetrahymena group I ribozyme's oligonucleotide substrate, CCCUCUA(5), f orms six base pairs with the ribozyme's internal guide sequence (IGS, 5'GGA GGG) to give the P1 duplex, and this duplex then docks into the active site via tertiary interactions. Shortening the substrate by three residues to g ive UCUA(5) reduces the equilibrium constant for P1 docking by similar to 2 00-fold even though UCUA(5) retains all the functional groups known to be i nvolved in tertiary interactions [Narlikar, G. J., Bartley, L. E., Khosla, M., and Herschlag, D. (1999) Biochemistry 38, 14192-14204]. Here we show th at the P1 duplex formed with UCUA5 engages in all of the major tertiary int eractions made by the standard P1 duplex. This suggests that the destabiliz ation is not due to disruption of specific tertiary interactions. It theref ore appears that the weaker docking of UCUA5 arises from the increased conf ormational freedom of the undocked 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 dupl ex with CCCUCUA(5)- but unpaired in the P1 duplex with UCUA(5) destabilizes docking of the standard P1 duplex similar to 300-fold more than it destabi lizes docking of the P1 duplex formed with UCUA(5). These results suggest t hat fixation of groups in the context of a rigid duplex may be a general st rategy used by RNA to substantially increase interaction specificity, both by aiding binding of the desired functional groups and by increasing the en ergetic cost of forming alternative interactions.