Dynamics of Large Elongated RNA by NMR Carbon Relaxation

摘要

We present an NMR strategy for characterizing picosecond-to-nanosecond internal motions in uniformly ~(13)C/~(15)N-labeled RNAs that combines measurements of R_1 R_(1p), and heteronuclear ~(13)C{~1H} NOEs for protonated base (C2, C5, C6, and C8) and sugar (C1') carbons with a domain elongation strategy for decoupling internal from overall motions and residual dipolar coupling (RDC) measurements for determining the average RNA global conformation and orientation of the principal axis of the axially symmetric rotational diffusion. TROSY-detected pulse sequences are presented for the accurate measurement of nucleobase carbon R_1 and R_(1p) rates in large RNAs. The relaxation data is analyzed using a model free formalism which takes into account the very high anisotropy of overall rotational diffusion (D_(ratop ≈ 4.7), asymmetry of the nucleobase CSAs and noncollinearity of C-C, C-H dipolar and CSA interactions under the assumption that all interaction tensors for a given carbon experience identical isotropic internal motions. The approach is demonstrated and validated on an elongated HIV-1 TAR RNA (τ_m ≈ 18 ns) both in free form and bound to the ligand argininamide (ARG). Results show that, while ARG binding reduces the amplitude of collective helix motions and local mobility at the binding pocket, it leads to a drastic increase in the local mobility of "spacer" bulge residues linking the two helices which undergo virtually unrestricted internal motions (S~2 ≈ 0.2) in the ARG bound state. Our results establish the ability to quantitatively study the dynamics of RNAs which are significantly larger and more anisotropic than customarily studied by NMR carbon relaxation.