MCA - The role of mRNA structure in allosteric control of ribosomal frameshifting

MCA - mRNA 结构在核糖体移码变构控制中的作用

• 批准号: 2122902
• 负责人: Peter Cornish
• 金额: $ 31.67万
• 依托单位: University of Missouri-Columbia
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2122902&HistoricalAwards=false
• 关键词: MCA; role; mRNA; structure; allosteric

Every protein in the cell is built from a sequence of amino acid building blocks by a large macromolecular machine: the ribosome. The specific arrangement of the amino acids in each protein is set as the ribosome reads the instructions, a message transcribed from DNA. Occasionally, the ribosome makes a mistake and slips out of register, changing the sequence of the product it manufactures. This slippage can result by chance, or it can be specifically engineered by the host. The latter results from a specific region within the message that causes slippage at the same place each time with a defined efficiency. The PI will investigate this mechanism of slippage with emphasis on how the ribosome steps through the manufacturing process, one amino acid at a time, and is made prone to slip. Further, the PI will develop methods to design novel sequences to either change the slippage frequency or to turn the process on or off. These fundamental details will help us understand how viruses exploit slippage during their reproduction. A molecular view of viral reproduction is key to the development of strategies to prevent viral transmission. As part of this project, the PI will develop a plan to enhance awareness of RNA biology through outreach efforts as well as provide research opportunities. The PI will strengthen outreach programs aimed at high-school aged youth in hopes of maintaining interest once these students reach a university. Protein synthesis is accomplished through a complex and highly coordinated process that requires a substantial amount of cellular energy. The central component in protein synthesis is the ribosome, which is a two-subunit macromolecular complex composed of both RNA and protein components. The ribosome binds to an mRNA molecule and faithfully translates the genetic code into an amino acid sequence. Large-scale conformational motions between and within its subunits facilitate this function. On occasion, translation fidelity can become compromised due to a failure of the ribosome. For example, the ribosome, when encountering specific structured sequences within the mRNA, can shift reading frames in the -1 direction instead of its normal forward progression of 3 nucleotides per amino acid incorporated. This suggests the hypothesis that aberrant changes in the conformation of the ribosome contribute to frameshifting. To investigate this process in detail, the following research will be performed: (i) determine the correlation between ribosomal conformational motions and -1 frameshifting using single molecule Förster resonance energy transfer (smFRET); (ii) determine the mechanism of RNA unwinding by the ribosome via smFRET and (iii) develop tailored and aptamer-controlled mRNA frameshifting elements by directed evolutionary approaches. Outcomes from these studies will be evaluated to provide a comprehensive understanding of -1 ribosomal frameshifting in both bacteria and eukaryotes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

细胞中的每种蛋白质都是由大型大分子机器：核糖体构建的。当核糖体读取指令时，设置了每种蛋白质中氨基酸的特定排列，这是从DNA转录的消息。有时，核糖体犯了一个错误并从注册中滑出，改变了其生产的产品的顺序。该滑台可以通过偶然产生，也可以由主机专门设计。后者是由于消息内的特定区域导致，该区域每次都以定义的效率在同一位置引起滑坡。 PI将研究这种滑动的机制，重点是核糖体如何在制造过程中逐步踏上一个氨基酸，并且容易滑动。此外，PI将开发设计新型序列以更改滑动频率或打开过程的方法。这些基本细节将有助于我们了解病毒在生殖过程中如何利用滑倒。病毒繁殖的分子视图是制定防止病毒传播策略的关键。作为该项目的一部分，PI将制定一项计划，通过外展工作以及提供研究机会来提高RNA生物学的认识。 PI将加强针对高中年龄的年轻人的外展计划，希望一旦这些学生进入大学，就可以保持兴趣。蛋白质合成是通过复杂且高度协调的过程来完成的，该过程需要大量的细胞能量。蛋白质合成中的中心成分是核糖体，它是由RNA和蛋白质成分组成的两征大分子复合物。核糖体与mRNA分子结合，并忠实地将遗传密码转化为氨基酸序列。其亚基之间和内部之间的大规模构象运动有助于此功能。有时，由于核糖体的失败，翻译忠诚会被妥协。例如，核糖体在mRNA中遇到特定的结构化序列时，可以在-1方向上移动读数框架，而不是其每个氨基酸掺入3个核动肽的正常正向进程。这表明核糖体构象的异常变化有助于射击。为了详细研究这一过程，将进行以下研究：（i）使用单分子försterförsterresonance能量传递（SMFRET）确定核糖体构象运动与-1帧的相关性； （ii）通过smfret确定核糖体解开RNA的机制，（iii）通过定向进化方法开发了量身定制的和置换器控制的mRNA frameshifting元件。这些研究的结果将得到评估，以全面了解细菌和真核生物中的-1核糖体框架。该奖项反映了NSF的法定任务，并通过使用基金会的智力优点和更广泛的影响标准来评估NSF的法定任务。