RNA: Structure, Biophysics and Physiology

RNA：结构、生物物理学和生理学

• 批准号: 10253855
• 负责人: Adrian Ferre-D'Amare
• 金额: $ 91.85万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10253855
• 关键词: Alternative Splicing; Anti-Bacterial Agents; Bacterial RNA; Biochemical; Biological Models; Biophysics; Catheters; Cell Aging; Cells; Chemicals; Clinical; Complement; Complex; DNA; DNA-Directed RNA Polymerase; Disease; Engineering; Folic Acid; Future; Gene Expression; Genetic Transcription; Health; Hybrids; Implant; Individual; Kinetics; Label; Length; Ligands; Location; Messenger RNA; Methods; Microbial Biofilms; Morbidity - disease rate; Nutritional; Physiological; Physiology; Polymerase; Procedures; Proteins; RNA; RNA Folding; Regulator Genes; Ribonucleoproteins; Spectrum Analysis; Stress; Structure; Study Subject; Technology; Telomerase; Thermodynamics; Time; Tissues; Translations; Vascular Endothelial Growth Factors; Work; helicase; interest; mRNA Decay; mortality; nutrition; response; single molecule; single-molecule FRET; small molecule; tumor growth; vector

In the past year we completed a single-molecule analysis of the mechanism of action of the ZTP riboswitch. This bacterial mRNA domain responds to ZTP, which accumulates during folate stress. Previous bulk and force-spectroscopy single-molecule studies have suggested that many riboswitches, especially those that function by regulating transcription, are under kinetic control, that is, function in a non-equilibrium regime. We studied a ZTP riboswitch by employing single-molecule FRET. Riboswitches that function transcriptionally typically fold in either a transcription-permissive, ligand-bound form, or default into a more thermodynamically stable intrinsic terminator fold. This ligand-concentration-dependent decision is made during transcription, as the RNA emerges from the polymerase and folds. Thus, to unravel the mechanism of action of such riboswitches, it is necessary to study them as they are made and fold. This has hitherto been impossible, because there no extant technology can introduce suitable labels onto an RNA as it emerges from the polymerase, without first pausing the polymerase (which introduces an artificial, protracted folding period). To overcome this limitation, we turned to an engineered helicase. We first prepared the full-length RNA, with labels introduced biochemically in ideal locations. Then, we hybridized this RNA to a full-length DNA complement. Finally, by addition of the helicase and ATP, we can release the RNA in the 5'-to-3' direction, analogous to how the RNA would emerge from the polymerase. We have employed two different helicases that allow us to explore rates of RNA release that correspond to fast- and slowly elongating bacterial RNA polymerase rates. Using this approach, we have, for the first time, produced single-molecule trajectories of a riboswitch as it folds directionally (or "vectorially") in the presence and absence of its cognate ligand. We find that the ZTP riboswitch is indeed under kinetic control, and that individual molecules occupy different channels in a complex regulatory landscape. More generally, our work shows that the control mechanism (kinetic vs thermodynamic) is intrinsic to the sequence of a riboswitch, and thus can be manifested regardless of whether the RNA folds co-transcriptionally, or is allowed to fold vectorially by other methods.

在过去的一年中，我们完成了ZTP Riboswitch作用机理的单分子分析。 该细菌mRNA结构域对ZTP有反应，ZTP在叶酸应激期间积累。 先前的体积和力谱镜单分子研究表明，许多核糖开关，尤其是那些通过调节转录功能的核糖开关在动力学控制下，即在非平衡状态下起作用。 我们通过采用单分子品格研究了ZTP Riboswitch。 在转录上发挥作用的核糖开关通常会在转录 - 允许的，结合配体形式或默认为热力学稳定的内在终端折叠中折叠。 在转录过程中，当RNA从聚合酶和褶皱中出现时，这是在转录过程中做出的。 因此，为了揭示这种核糖开关的作用机理，有必要在制造并折叠时研究它们。 迄今为止，这是不可能的，因为没有现有的技术可以将合适的标签从聚合酶出现时引入RNA，而不会先暂停聚合酶（该聚合酶引入了人工，持久的折叠周期）。 为了克服这一限制，我们转向了工程化的解旋酶。 我们首先准备了全长RNA，标签在理想位置引入了生化。 然后，我们将此RNA杂交到全长DNA补体。 最后，通过添加解旋酶和ATP，我们可以沿5'至3'方向释放RNA，类似于RNA如何从聚合酶中出现。 我们采用了两种不同的解旋酶，使我们能够探索与快速和缓慢拉长细菌RNA聚合酶速率相对应的RNA释放速率。 使用这种方法，我们首次产生了核糖开关的单分子轨迹，因为它在存在和不存在其同源配体的情况下会折叠（或“矢量上”）。 我们发现ZTP核糖开关确实处于动力学控制之下，并且单个分子在复杂的调节景观中占据了不同的通道。 更普遍地，我们的工作表明，控制机制（动力学与热力学）是核糖开关的序列固有的，因此无论RNA折叠是共转录的，还是可以通过其他方法允许向量折叠。