Graduate Program in Biochemistry and Biophysics, Rensselaer Polytechnic Institute, Troy, NY 12180.
Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180.
Proc Natl Acad Sci U S A. 2023 Jun 27;120(26):e2215556120. doi: 10.1073/pnas.2215556120. Epub 2023 Jun 20.
Conformational dynamics play essential roles in RNA function. However, detailed structural characterization of excited states of RNA remains challenging. Here, we apply high hydrostatic pressure (HP) to populate excited conformational states of tRNA, and structurally characterize them using a combination of HP 2D-NMR, HP-SAXS (HP-small-angle X-ray scattering), and computational modeling. HP-NMR revealed that pressure disrupts the interactions of the imino protons of the uridine and guanosine U-A and G-C base pairs of tRNA. HP-SAXS profiles showed a change in shape, but no change in overall extension of the transfer RNA (tRNA) at HP. Configurations extracted from computational ensemble modeling of HP-SAXS profiles were consistent with the NMR results, exhibiting significant disruptions to the acceptor stem, the anticodon stem, and the D-stem regions at HP. We propose that initiation of reverse transcription of HIV RNA could make use of one or more of these excited states.
构象动力学在 RNA 功能中起着至关重要的作用。然而，详细的 RNA 激发态结构表征仍然具有挑战性。在这里，我们应用高静水压力 (HP) 使 tRNA 进入激发构象状态，并使用 HP 二维 NMR、HP-SAXS（高压力小角 X 射线散射）和计算建模相结合对其进行结构表征。HP-NMR 表明压力破坏了 tRNA 中尿嘧啶和鸟嘌呤 U-A 和 G-C 碱基对的亚氨基质子的相互作用。HP-SAXS 图谱显示形状发生了变化，但在 HP 下 tRNA（转移 RNA）的整体延伸没有变化。从 HP-SAXS 图谱的计算整体建模中提取的构象与 NMR 结果一致，在 HP 下，对接受茎、反密码子茎和 D-茎区域有明显的破坏。我们提出，HIV RNA 逆转录的起始可能利用这些激发态中的一个或多个。
