Resolving the Motional Modes That Code for RNA Adaptation

Abstract

Using a domain elongation strategy, we decoupled internal motions in RNA from overall rotational diffusion. This allowed us to site-specifically resolve a manifold of motional modes in two regulatory RNAs from HIV-1 with the use of nuclear magnetic resonance spin relaxation methods. Base and sugar librations vary on a picosecond time scale and occur within helical domains that move collectively at diffusion-limited nanosecond time scales. Pivot points are short, functionally important, and highly mobile internal loops. These spontaneous changes in RNA conformation correlate quantitatively with those that follow adaptive recognition of diverse targets. Thus, ligands may stabilize existing RNA conformations rather than inducing new ones.

HIV-1 TAR RNA Spontaneously Undergoes Relevant Apo-to-Holo Conformational Transitions in Molecular Dynamics and Constrained Geometrical Simulations

“…These above results are consistent with the experimental view that the bound TAR RNA is stabilized by weak intermolecular interactions with the ligand 27 and that bound conformations are transiently sampled in the free ensemble, 4,5 following the conformation selection model. The results are also encouraging from an application point of view in that those simulated TAR RNA conformations can be used as receptor structures for docking.…”

“…Notably, these numbers are of the same order of magnitude as, but smaller than, the binding free energies of the respective ligands such that weak intermolecular interactions with the ligands should suffice to stabilize bound TAR RNA. 27 Furthermore, the small conformational free energy differences are consistent with the view that bound TAR conformations are transiently sampled in the free ensemble, 4,5 following the conformation selection model.…”

“…In this regard, one of the best investigated RNA structures is the HIV-1 transactivation response element (TAR). [2][3][4][5][6] TAR RNA is a 59-nucleotide hairpin structure located at the 5′-end of all pre-mRNA in a HIV-1 transcript. Binding of the regulatory Tat protein to a bulge region within the TAR RNA structure is an essential step for HIV-1 viral replication.…”

NMR studies of dynamics in RNA and DNA by 13C relaxation

“…This operation decouples the time scale of reorientation of the two helical elements with respect to one another from the global tumbling of the molecule. 68 The key to successful execution resides in preparing two samples, each containing only one type of base pairs in the elongation helix (Gua and Cyt or Ade and Ura) and introducing complementary isotope-labels (AU or GC) to make the elongated RNA invisible. The intensity of 13 C resonances in the helical stems of the full-length and elongated samples can be compared to determine in a straightforward way if domain motions exist.…”

“…In addition, elongation studies of HIV-1 TAR determined that the upper helix moves as a single rigid unit on a nanosecond timescale, across the U23-U25 bulge. 68 Human U1A protein regulates it own production by binding to the 3 0 untranslated region of its own pre-mRNA by undergoing a conformational change that allows U1A to bind to poly(A)-polymerase enzyme and inhibits polyadenylation. 78-80 13 C relaxation and power dependence studies of the free RNA demonstrated that residues in the binding region experienced motion on multiple timescales (ns-ps, s-ms).…”