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

细胞中的每一种蛋白质都是由一个大的大分子机器--核糖体--由一系列氨基酸组成的。每种蛋白质中氨基酸的特定排列是在核糖体阅读指令时设定的，指令是从DNA转录而来的信息。偶尔，核糖体会犯错误，滑出注册表，改变其生产的产品的序列。这种滑移可能是偶然的，也可能是主人特意设计的。后者是由消息内的特定区域引起的，该区域每次以定义的效率在相同的位置造成滑移。PI将研究这种滑动的机制，重点是核糖体如何通过制造过程，一次一个氨基酸，并使其容易滑动。此外，PI将开发设计新序列的方法，以改变滑动频率或开启或关闭该过程。这些基本的细节将帮助我们理解病毒是如何利用其繁殖过程中的滑动的。病毒复制的分子观点是制定预防病毒传播策略的关键。作为该项目的一部分，国际和平研究所将制定一项计划，通过外展努力提高对RNA生物学的认识，并提供研究机会。PI将加强针对高中年长青年的外联计划，希望这些学生一旦进入大学就能保持兴趣。蛋白质的合成是通过一个复杂和高度协调的过程来完成的，这需要大量的细胞能量。蛋白质合成的中心成分是核糖体，它是由RNA和蛋白质组成的两个亚单位的大分子复合体。核糖体与信使核糖核酸分子结合，将遗传密码忠实地翻译成氨基酸序列。亚基之间和亚基内的大规模构象运动促进了这一功能。有时，由于核糖体的故障，翻译的保真度可能会受到影响。例如，当核糖体遇到mRNA中的特定结构序列时，可以沿-1方向移动阅读框，而不是其正常的向前推进，即每掺入一个氨基酸3个核苷酸。这表明，核糖体构象的异常变化有助于移码的假说。为了深入研究这一过程，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移框之间的相关性；(Ii)确定RNA通过smFRET由核糖体解旋的机制；以及(Iii)通过定向进化方法开发定制的和适体控制的mRNA移框元件。将对这些研究的结果进行评估，以提供对细菌和真核生物中核糖体移码的全面了解。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.