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

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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.