Antagonistic Substrate Binding by a Group II Intron Ribozyme

J Mol Biol. 1999 Aug 6;291(1):15-27. doi: 10.1006/jmbi.1999.2922.

Abstract

In this study, the thermodynamic properties of substrate-ribozyme recognition were explored using a system derived from group II intron ai5gamma. Substrate recognition by group II intron ribozymes is of interest because any nucleic ac?id sequence can be targeted, the recognition sequence can be quite long (>/=13 bp), and reaction can proceed with a very high degree of sequence specificity. Group II introns target their substrates 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 sites (IBS1 and IBS2) on the substrate. In order to understand the relative energetic contribution of each base-pairing interaction (EBS1-IBS1 or EBS2-IBS2) to substrate binding energy, the free energy of each helix was measured. The individual helices were found to have base-pairing free energies similar to those calculated for regular RNA duplexes of the same sequence, suggesting that each recognition helix derives its binding energy from base-pairing interactions alone and that each helix can form independently. Most interestingly, it was found that the sum of the measured individual free energies (approximately 20 kcal/mol) was much higher than the known free energy for substrate binding (approximately 12 kcal/mol). This indicates that certain group II intron ribozymes can bind their substrates in an antagonistic fashion, 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 substrate binding. This coupling between substrate binding and ribozyme conformational rearrangement may provide a mechanism for lowering overall substrate binding energy while retaining the full information content of 13 bp, thus resulting in a mechanism for ensuring sequence specificity.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Binding Sites
  • Catalysis
  • Introns*
  • Mutation
  • Nucleic Acid Conformation
  • RNA Splicing
  • RNA, Catalytic / antagonists & inhibitors
  • RNA, Catalytic / chemistry
  • RNA, Catalytic / metabolism*
  • Substrate Specificity
  • Thermodynamics

Substances

  • RNA, Catalytic