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.

去年我们完成了 ZTP 核糖开关作用机制的单分子分析。 该细菌 mRNA 结构域对 ZTP 做出反应，ZTP 在叶酸应激期间积累。 先前的体积和力谱单分子研究表明，许多核糖开关，尤其是那些通过调节转录发挥作用的核糖开关，处于动力学控制之下，即在非平衡状态下发挥作用。 我们利用单分子 FRET 研究了 ZTP 核糖开关。 转录功能的核糖开关通常以转录许可、配体结合的形式折叠，或者默认为热力学更稳定的内在终止子折叠。 当 RNA 从聚合酶中出现并折叠时，这种配体浓度依赖性决定是在转录过程中做出的。 因此，为了揭示此类核糖开关的作用机制，有必要研究它们的制造和折叠过程。 迄今为止，这是不可能的，因为没有现有的技术可以在RNA从聚合酶中出现时将合适的标记引入到RNA上，而无需首先暂停聚合酶（这会引入人为的、延长的折叠期）。 为了克服这个限制，我们转向了工程解旋酶。 我们首先制备全长 RNA，并在理想位置通过生化方法引入标签。 然后，我们将该 RNA 与全长 DNA 互补体杂交。 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 从聚合酶中出现的方式。 我们使用了两种不同的解旋酶，使我们能够探索与快速和缓慢延伸细菌 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在其同源配体存在和不存在的情况下定向（或“矢量”）折叠的单分子轨迹。 我们发现 ZTP 核糖开关确实受到动力学控制，并且各个分子在复杂的调控环境中占据不同的通道。 更一般地说，我们的工作表明，控制机制（动力学与热力学）是核糖开关序列所固有的，因此无论RNA是否共转录折叠，或是否允许通过其他方法矢量折叠，都可以表现出来。