Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Strobel, Scott

Abstract

Protein synthesis is both essential and highly regulated. This process is complex and requires the concerted effort of many protein factors. Some viruses have evolved ways to co-opt host translation during initiation by using internal ribosome entry sites (IRESs). These structured RNAs allow the virus to bind to the host's ribosome in the absence of canonical requirements. This dissertation focuses on relating the sequence of viral IRES elements to their role in initiating translation. The Cricket Paralysis Virus (CrPV) IRES is short, uses a simple mechanism, and can function in diverse cell-free systems making it a useful tool to study the mechanism of translation and to express proteins. I used a RelE-based next-generation sequencing method, termed SMARTI, to quantitatively determine the function of over 81,000 single and double mutants of CrPV IRES. I then applied this high-throughput method to two other IRES sequences, Pso IRES and HalV IRES, to determine which sequence elements are driving their unique binding abilities. In doing so, I produced quantitative ribosome binding data for 98,346 and 84,491 mutants respectively. The result of these studies is a comprehensive mutational database that serves as a consensus sequence-like analysis of IRES function. Further, I demonstrated that this high-throughput method is compatible with eukaryotic extract. Together, my work provides an avenue for studying diverse eukaryotic RNA elements, designing therapeutics, and for engineering sequences for protein expression.

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