Characterizing Complex Dynamics in the Transactivation Response Element Apical Loop and Motional Correlations with the Bulge by NMR, Molecular Dynamics, and Mutagenesis

• Published in: Biophysical journal 2008-10, Vol.95 (8), p.3906-3915
• Main Authors: Dethoff, Elizabeth A., Hansen, Alexandar L., Musselman, Catherine, Watt, Eric D., Andricioaei, Ioan, Al-Hashimi, Hashim M.
• Format: Article
• Language: English
• Subjects: Base Sequence; Binding sites; Biophysics; Carbon Isotopes; Coupling (molecular); Dispersions; Dynamic tests; Dynamics; HIV-1 - chemistry; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular biology; Molecular dynamics; Molecular Sequence Data; Mutagenesis; Mutation - genetics; Nitrogen Isotopes; Nuclear magnetic resonance; Nucleic Acid Conformation; Nucleic Acids; Recognition; Response Elements - genetics; Ribonucleic acid; Ribonucleic acids; RNA; RNA, Viral - chemistry; RNA, Viral - genetics; Transcriptional Activation - genetics
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1529/biophysj.108.140285

The HIV-1 transactivation response element (TAR) RNA binds a variety of proteins and is a target for developing anti-HIV therapies. TAR has two primary binding sites: a UCU bulge and a CUGGGA apical loop. We used NMR residual dipolar couplings, carbon spin relaxation (R1 and R2), and relaxation dispersion (R1ρ) in conjunction with molecular dynamics and mutagenesis to characterize the dynamics of the TAR apical loop and investigate previously proposed long-range interactions with the distant bulge. Replacement of the wild-type apical loop with a UUCG loop did not significantly affect the structural dynamics at the bulge, indicating that the apical loop and the bulge act largely as independent dynamical recognition centers. The apical loop undergoes complex dynamics at multiple timescales that are likely important for adaptive recognition: U31 and G33 undergo limited motions, G32 is highly flexible at picosecond-nanosecond timescales, and G34 and C30 form a dynamic Watson-Crick basepair in which G34 and A35 undergo a slow (∼30μs) likely concerted looping in and out motion, with A35 also undergoing large amplitude motions at picosecond-nanosecond timescales. Our study highlights the power of combining NMR, molecular dynamics, and mutagenesis in characterizing RNA dynamics.