Dethoff Elizabeth A, Hansen Alexandar L, Musselman Catherine, Watt Eric D, Andricioaei Ioan, Al-Hashimi Hashim M
Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Biophys J. 2008 Oct;95(8):3906-15. doi: 10.1529/biophysj.108.140285. Epub 2008 Jul 11.
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 (R(1) and R(2)), and relaxation dispersion (R(1rho)) 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 (approximately 30 mus) 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.
HIV-1反式激活应答元件（TAR）RNA可结合多种蛋白质，是开发抗HIV疗法的一个靶点。TAR有两个主要结合位点：一个UCU凸起和一个CUGGGA顶端环。我们结合分子动力学和诱变技术，利用核磁共振剩余偶极耦合、碳自旋弛豫（R(1)和R(2)）以及弛豫色散（R(1rho)）来表征TAR顶端环的动力学，并研究先前提出的与远处凸起的长程相互作用。用UUCG环取代野生型顶端环对凸起处的结构动力学没有显著影响，这表明顶端环和凸起在很大程度上作为独立的动态识别中心起作用。顶端环在多个时间尺度上经历复杂的动力学过程，这可能对适应性识别很重要：U31和G33的运动受限，G32在皮秒到纳秒的时间尺度上高度灵活，G34和C30形成一个动态的沃森-克里克碱基对，其中G34和A35经历缓慢（约30微秒）的可能协同的进出环运动，A35在皮秒到纳秒的时间尺度上也经历大幅度运动。我们的研究突出了结合核磁共振、分子动力学和诱变技术在表征RNA动力学方面的作用。
