Helix-junction-helix (HJH) motifs are flexible building blocks of RNA architecture that help define the orientation and dynamics of helical domains. They are also frequently involved in adaptive recognition of proteins and small molecules, and in the formation of tertiary contacts. Here, we use a battery of nuclear magnetic resonance (NMR) techniques to examine how deleting a single bulge residue (C24) from the human immunodeficiency virus type 1 (HIV-1) transactivation response element (TAR) tri-nucleotide bulge (U23-C24-U25) affects dynamics over a broad range of timescales. Shortening the bulge has a similar effect on picosecond-to-nanosecond inter-helical and local bulge dynamics, as increasing the Mg(2+) and Na(+) concentration; whereby a pre-existing two-state equilibrium in TAR is shifted away from a bent flexible conformation towards a coaxial conformation, in which all three bulge residues are flipped out and flexible. Surprisingly, the point deletion minimally affects microsecond-to-millisecond conformational exchange directed toward two low-populated and short-lived excited conformational states that form through reshuffling of bases pairs throughout TAR. The mutant does, however, adopt a slightly different excited conformational state on the millisecond timescale, in which U23 is intra-helical, mimicking the expected conformation of residue C24 in the excited conformational state of wild-type TAR. Thus, minor changes in HJH topology preserve motional modes in RNA occurring over the picosecond-to-millisecond timescales, but alter the relative populations of the sampled states or cause subtle changes in their conformational features.
Shortening the HIV-1 TAR RNA Bulge by a Single Nucleotide Preserves Motional Modes Over a Broad Range of Timescales.