Cooperative and anticooperative binding to a ribozyme

Abstract

The effects of guanosine 5′-monophosphate and 2′-deoxyguanosine 5′-monophosphate on the thermodynamics and kinetics of pyrene-labeled 5′ exon mimic (pyCUCU) binding to the catalytic RNA (ribozyme) from Tetrahymena thermophila have been determined by fluorescence titration and kinetics experiments at 15°C. pyCUCU binding to L-21 Sca I-truncated ribozyme is weaker by a factor of 5 in the presence of saturating guanosine 5′-monophosphate, whereas it is 4-fold stronger in the presence of saturating 2′-deoxyguanosine 5′-monophosphate. Results from kinetics experiments suggest that ant kooperative effects in the presence of guanosine 5′-monophosphate arise primarily from slower formation of tertiary contacts between the catalytic core of the ribozyme and the P1 duplex formed by pyCUCU and GGAGGG of the ribozyme. Conversely, cooperative effects in the presence of 2′-deoxyguanosine 5′-monophosphate arise primarily from slower disruption of tertiary contacts between the catalytic core of the ribozyme and the P1 duplex. Additional experiments suggest that these cooperative and anticooperative effects are not a function of the pyrene label, are not caused by a salt effect, and are not specific to one renaturation procedure for the ribozyme.