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.

细胞中的每一种蛋白质都是由一个巨大的大分子机器——核糖体——由一系列氨基酸组成的。当核糖体读取从DNA转录而来的指令时，每种蛋白质中氨基酸的特定排列就被确定了。偶尔，核糖体犯了一个错误，滑出了寄存器，改变了它制造的产品的序列。这种滑移可能是偶然造成的，也可能是由宿主专门设计的。后者来自消息中的特定区域，每次在同一位置以定义的效率导致滑动。PI将研究这种滑移的机制，重点是核糖体如何通过制造过程，一次一个氨基酸，并使其易于滑移。此外，PI将开发方法来设计新的序列，以改变滑动频率或打开或关闭过程。这些基本细节将帮助我们了解病毒在繁殖过程中是如何利用滑脱的。病毒复制的分子观点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，通过外展工作提高对RNA生物学的认识，并提供研究机会。PI将加强针对高中青少年的外展计划，希望这些学生进入大学后能保持兴趣。蛋白质合成是一个复杂而高度协调的过程，需要大量的细胞能量。蛋白质合成的核心成分是核糖体，它是一种由RNA和蛋白质组分组成的双亚基大分子复合物。核糖体与mRNA分子结合，忠实地将遗传密码翻译成氨基酸序列。其亚基之间和内部的大规模构象运动促进了这一功能。有时，由于核糖体的失效，翻译的保真度可能会受到损害。例如，当核糖体在mRNA中遇到特定的结构序列时，可以将阅读框向-1方向移动，而不是正常的每个氨基酸向前移动3个核苷酸。这表明核糖体构象的异常变化有助于移框的假设。为了详细研究这一过程，将进行以下研究：(i)利用单分子Förster共振能量转移（smFRET）确定核糖体构象运动与-1移帧之间的相关性；（ii）通过smFRET确定核糖体解绕RNA的机制；（iii）通过定向进化方法开发定制的、由适体控制的mRNA移框元件。这些研究的结果将被评估，以提供对细菌和真核生物-1核糖体移框的全面理解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。