Shortening the HIV-1 TAR RNA Bulge by a Single Nucleotide Preserves Motional Modes over a Broad Range of Time Scales

Abstract

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 techniques to examine how deleting a single bulge residue (C24) from the human immunodeficiency virus type 1 (HIV-1) transactivation response element (TAR) trinucleotide bulge (U23-C24-U25) affects dynamics over a broad range of time scales. Shortening the bulge has an effect on picosecond-to-nanosecond interhelical and local bulge dynamics similar to that casued by increasing the Mg2+ and Na+ concentration, whereby a preexisting two-state equilibrium in TAR is shifted away from a bent flexible conformation toward 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 time scale, in which U23 is intrahelical, 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 time scales but alter the relative populations of the sampled states or cause subtle changes in their conformational features.

Probing RNA Conformational Equilibria within the Functional Cellular Context

“…Likewise, the partial rescue of TAR-G36U/ C29A in the transactivation assay mirrors the partial conformational rescue as assessed by NMR ( Figures 2C and 2D), although we cannot rule out that the identity of the closing G36-C29 base pair is also important for protein recognition ( Figures 2C and 2D). The line broadening should, however, be interpreted cautiously, since it could also reflect at least in part dimerization of the ES2 conformation, as reported previously (Merriman et al, 2016). Based on NMR, the rescue mutants TAR-U38A/A27U and TAR-A27C/U38G strongly stabilize the GS, yet they only partially restore activity ( Figures 2C and 2D).…”

“…A previous study found that the ES2-stabilizing mutant, TAR-G28U, experiences a small amount of dimerization at NMR concentrations and two imino peaks were assigned to the inter-molecular U31-G32 bp (Merriman et al, 2016). Evaluation of the other TAR mutants using 1D NMR of the imino protons reveal that the other ES2-stabilizing mutants and, to a lesser extent, rescue mutants also have minor peaks corresponding to dimerization ( Figures S3A and S3B).…”

Atomic structures of excited state A–T Hoogsteen base pairs in duplex DNA by combining NMR relaxation dispersion, mutagenesis, and chemical shift calculations

“…Our study demonstrates that a mutational approach can also be valid in the case of nucleic acids. Indeed, we previously showed that point substitution mutations as well as single-atom substitutions can be used to stabilize RNA ESs 8,16,17,74 . In addition, various forms of mutagenesis have successfully been used to stabilize ESs of proteins 30,33,34,75,76 .…”

RNA Dynamics by NMR Spectroscopy

“…Furthermore, ap roperc hemical shift database and denovo chemical shift prediction, for example, by using DFT,w ill simplify the elucidation of excited states, which is currently te-dious and labor intensive. Additionally,f ew studies currently exist where systems have been compared across experiments and timescales, for example, [43,108,136] which is an important part to expand on in the future.…”

“…Systems studied on fast timescale have been recently reviewed by Ban et al, [105] Bardon et al [106] and Rinnenthal et al [107] One of the latest examples where many timescales were covered,i st he fluoride riboswitch (Figure 20), for which the ligand-bound state and free state exhibit similar dynamic profiles. [43] Recent studies with similar experimental setup, mostly probing 1 H, 13 Ca nd sometimes 1 H, 15 Nv ectorsh ave been published on TARa nd modifications thereof, [108,109] SOLE RNA, [110] apicall oop HBV RNA, [2,111] single-stranded RNA part of the prequeuosine riboswitch, [112,113] pseudoknot of the telomerase RNA [114] and the well-known UUCG tetraloop, where 31 Pd ynamics were probed. [115] An interesting applicationi st he estimation of TARR NA binding to dendrimers using 13 Cs pin-relaxation rates.…”