Site-directed spin labeling studies reveal solution conformational changes in a GAAA tetraloop receptor upon Mg(2+)-dependent docking of a GAAA tetraloop.

Abstract

The Mg(2+)-dependent GAAA tetraloop interaction with its 11 nucleotide receptor is one of the most frequently occurring long-range tertiary interactions in RNAs. To explore conformational changes in the receptor during tetraloop docking, nitroxide spin labels were attached at each of four uridine bases, one at a time, within an RNA molecule containing the receptor sequence. In the presence of Mg2+ and the tetraloop, the electron paramagnetic resonance (EPR) spectrum of one of the labeled bases reflected a large increase in mobility, indicating unstacking of the base upon tetraloop docking. This provides direct evidence that base unstacking is an intrinsic feature of the solution tetraloop-receptor complex formed in the presence of Mg2+. Additional evidence suggests that in solution the bound receptor conformation is similar to that observed in the crystal structure of a group I intron ribozyme domain. In Mg2+ alone, a receptor conformation with an unstacked base was not detectable, suggesting that this conformation is of higher standard state free energy than that of the free receptor. This leads to the conclusion that the extensive RNA-RNA interactions observed in the crystal structure of the tetraloop-receptor complex provide larger interaction energy than the measured apparent affinity between the tetraloop and the free receptor. This is compatible with a high specificity of the tetraloop-receptor interaction.