Abstract
The HIV-1 Rev response element (RRE) RNA element mediates the nuclear export of intron containing viral RNAs by forming an oligomeric complex with the viral protein Rev. Stem IIB and nearby stem II three-way junction nucleate oligomerization through cooperative binding of two Rev molecules. Conformational flexibility at this RRE region has been shown to be important for Rev binding. However, the nature of the flexibility has remained elusive. Here, using NMR relaxation dispersion, including a new strategy for directly observing transient conformational states in large RNAs, we find that stem IIB alone or when part of the larger RREII three-way junction robustly exists in dynamic equilibrium with non-native excited state (ES) conformations that have a combined population of ∼20%. The ESs disrupt the Rev-binding site by changing local secondary structure, and their stabilization via point substitution mutations decreases the binding affinity to the Rev arginine-rich motif (ARM) by 15- to 80-fold. The ensemble clarifies the conformational flexibility observed in stem IIB, reveals long-range conformational coupling between stem IIB and the three-way junction that may play roles in cooperative Rev binding, and also identifies non-native RRE conformational states as new targets for the development of anti-HIV therapeutics.
Highlights
RNAs are growing in their importance as regulators of gene expression (1-3), novel drug targets (4-6), and as tools for bioengineering applications and synthetic biology [7,8]
These results indicate that the three-way junction and Mg2+ have small effects on the ground state (GS) conformation but do alter micro-to-millisecond conformational exchange at the internal loop region
Our study adds to a growing view that non-canonical regions of RNA do not fold into a single secondary structure but rather exist as a dynamic equilibrium of alternative conformations that have non-native secondary structure (16-19)
Summary
RNAs are growing in their importance as regulators of gene expression (1-3), novel drug targets (4-6), and as tools for bioengineering applications and synthetic biology [7,8]. RNA and other biomolecules do not fold into a single structure but rather form a statistical ensemble of many interconverting conformations (9-12). Binding of ligands, proteins, other RNAs, or changes in physiological conditions such as temperature can favor specific conformations in the ensemble and cause a structure specific change in activity (13-15). A deep understanding of how RNAs function within cells requires an understanding of their dynamic behavior. This in turn may enable the targeting of RNA in drug discovery efforts [12,13] as well as the rational design of RNA-based devices [7]
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