Antagonistic substrate binding by a group II intron ribozyme

Ln this study, the thermodynamic properties of substrate-ribozyme recogniti on were explored using a system derived from group II intron ai5 gamma. Sub strate recognition by group II intron ribozymes is of interest because any nucleic ac?id sequence can he targeted, the recognition sequence can be qui te long (greater than or equal to 13 bp), and reaction can proceed with a v ery high degree of sequence specificity. Group II introns target their subs trates throug?h the formation of base-pairing interactions with two regions of the intron (EBS1 and EBS2), which are usually located far apart in the secondary structure. These structures pair with adjacent, corresponding sit es (IBS1 and IBS2) on the substrate. Ln order to understand the relative en ergetic contribution of each base-pairing interaction (EBS1-IBS1 or EBS2-IB S2) to substrate binding energy, the free energy of each helix was measured . The individual helices were found to have base-pairing free energies simi lar to those calculated for regular RNA duplexes of the same sequence, sugg esting that each recognition helix derives its binding energy from base-pai ring interactions alone and that each helix can form independently. Most in terestingly, it was found that the sum of the measured individual free ener gies (similar to 20 kcal/mol) was much higher than the known free energy fo r substrate binding (similar to 12 kcal/mol). This indicates that certain g roup II intron ribozymes can bind their substrates in an antagonistic fashi on, paying a net energetic penalty upon binding the full-length substrate. This loss of binding energy is not due to weakening of individual helices, but appears to be linked to ribozyme conformational changes induced by subs trate binding. This coupling between substrate binding and ribozyme conform ational rearrangement may provide a mechanism for lowering overall substrat e binding energy while retaining the full information content of 13 bp, thu s resulting in a mechanism for ensuring sequence specificity. (C) 1999 Acad emic Press.