Stopped-Flow Fluorescence Spectroscopy of a Group II Intron Ribozyme Reveals that Domain 1 Is an Independent Folding Unit with a Requirement for Specific Mg2+ Ions in the Tertiary Structure

Abstract

A stopped-flow fluorescence spectroscopic assay for RNA folding was used to monitor the association of a multicomponent ribozyme derived from group II intron ai5γ. In the presence of Mg²⁺, association of a short fluorescein-labeled oligonucleotide substrate with intron domain 1 (D1) resulted in a unique fluorescein emission enhancement, which reflected ribozyme folding and substrate binding. It was found that substrate binding follows a simple bimolecular encounter model, with k_on approaching the rate of simple duplex formation. The K_d between substrate and D1 was determined to be 11 nM, which is in close agreement with the K_d obtained through oligonucleotide cleavage assays requiring catalytic domain 5 (D5). Ribozyme variants D13 and D135, which contain D3 and/or D5 attached to D1 in-cis, bound substrate with very similar K_d values, suggesting that D1 can fold independently and contains all residues important for ground-state binding to substrate. Both stopped-flow fluorescence assays and chemical modification footprinting data showed that, in all three ribozymes, Mg²⁺ was required and sufficient for folding. The rates of substrate association and the fraction of active ribozyme showed similar [Mg²⁺] dependencies, indicating that folding and substrate binding in these three ribozymes are the result of similar processes involving specific, weakly bound Mg²⁺ ions. The apparent binding constants for the Mg²⁺ ions were found to be approximately 70 mM in each case. Together, these data show that D1 is an independent folding unit with respect to substrate binding and that specific Mg²⁺ ions are required for the formation of a distinct tertiary structure in group II introns.