Single-molecule folding studies and genetic regulation of a S-adenosylmethionine (SAM) riboswitch / Études de la régulation génétique et du repliement du ribogétulateur SAM

S-腺苷甲硫氨酸 (SAM) 核糖开关的单分子折叠研究和遗传调控 / Átudes de la régulation génétique et du repliement du ribogétulateur SAM

• 批准号: 262090-2013
• 负责人: Lafontaine, Daniel
• 金额: $ 4.23万
• 依托单位: Université de Sherbrooke
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=571233
• 关键词: Single; molecule; folding; studies; genetic

Riboswitches are structured motifs that usually reside in the non-coding region of mRNAs, where they bind metabolites and control gene expression. Unlike many other genetic control systems, riboswitches do not directly require metabolite-binding proteins to act as sensors, and thus provide a direct link between the genetic information that is encoded by an mRNA and its cellular environment. This research program will first aim to characterize the importance of residues of the S-adenosylmethionine (SAM) aptamer that are required to attain the native structure using a variety of biochemical and biophysical techniques, such as FRET (Fluorescence Resonance Energy Transfer). FRET provides a unique way of determining angles between subtended different arms in a junction and the identification of stacking partners. Key mutants will be engineered to study how riboswitch folding domains are used in the ligand recognition mechanism. The second goal will be the characterization of a recently uncovered folding transition, the ligand-dependent helical twist. The third goal of the proposed research will consist in the characterization of the riboswitch conformational changes in real time, which is only attainable using single-molecule approaches. The single-molecule FRET (sm-FRET) technique is unique in its ability to monitor dynamic structural changes of RNA molecules in real time, which provides a complete view of the folding pathway of riboswitches by revealing discrete intermediates together with their associated folding rate(s). Such measurements will be obtained using a state-of-the-art single-molecule total-internal reflection (TIR) setup coupled to an electron-multiplying CCD camera allowing observation time in the range of the low-millisecond timescale. Clearly, we expect to characterize the entire folding pathway of the riboswitch and to obtain a more complete understanding by which ligand binding to the riboswitch is harnessed to ensure a timely and efficient genetic regulation. The characterization of the SAM riboswitch will provide new insights on RNA folding mechanisms, and how it is involved in the ligand binding specificity that is crucial for the control of genetic expression.

核糖开关是一种结构化的基序，通常位于mRNA的非编码区，在那里它们结合代谢物并控制基因表达。与许多其他遗传控制系统不同，核糖开关不直接需要代谢物结合蛋白作为传感器，因此在mRNA编码的遗传信息与其细胞环境之间提供了直接联系。该研究计划的目的首先是表征S-腺苷甲硫氨酸（SAM）适体残基的重要性，这些残基是使用各种生物化学和生物物理技术（如FRET）获得天然结构所需的。FRET提供了一种独特的方式来确定对向不同的臂之间的角度在一个路口和堆叠合作伙伴的识别。关键突变体将被工程化以研究核糖开关折叠结构域如何用于配体识别机制。第二个目标将是表征最近发现的折叠过渡，配体依赖的螺旋扭曲。所提出的研究的第三个目标将包括核糖开关构象变化的表征在真实的时间，这是只能实现使用单分子的方法。单分子FRET（sm-FRET）技术能够真实的实时监测RNA分子的动态结构变化，通过揭示离散的中间体及其相关的折叠速率，提供了核糖开关折叠途径的完整视图。这样的测量将获得使用一个国家的最先进的单分子全内反射（TIR）设置耦合到电子倍增CCD相机，允许在低毫秒的时间尺度范围内的观察时间。显然，我们期望表征核糖开关的整个折叠途径，并获得更完整的理解，其中配体结合核糖开关是利用，以确保及时和有效的遗传调控。SAM核糖开关的表征将为RNA折叠机制以及它如何参与对基因表达控制至关重要的配体结合特异性提供新的见解。