Genetic screen for highly active variants of the B. subtilis pbuE adenine-responsive riboswitch

Kim, Mia B.

Undergraduate Honors Thesis

Genetic screen for highly active variants of the B. subtilis pbuE adenine-responsive riboswitch Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/pg15bg088

Abstract

Riboswitches are sequences found in the 5’ untranslated region (5’ UTR) of mRNAs that controls downstream gene expression by binding specific small molecules. This binding event allows for a turn on or off expression in response. Riboswitches are composed of two domains, the aptamer domain, and the expression platform. The aptamer domain acts as a receptor that binds a ligand. The expression platform directly influences gene expression though its ability to switch between two different secondary structures in response to ligand binding. Riboswitches are important because they are endogenous machinery in bacteria and in some archaea that regulate both transcription and translation. This makes them potentially desirable for the development of pharmacological targets or as biosensors that could be deployed for field work to check for certain molecules in samples. The B. subtilis pbuE riboswitch binds to adenine and its analog, 2-aminopurine, to regulate gene expression of the downstream mRNA. In this research project, I performed a survey that focused on key elements of the expression platform, crucial in the formation of the terminator to understand nucleotide sequences important for efficient switching. To do so, two regions of a hairpin stem-loop in the expression platform were randomized: the loop library and the stem library.

From the observed results, there was no clear sequence consensus in the loop region of the expression platform, and most switches performed comparably to parental riboswitch, indicating that specificity in that region is low. However, in the stem library that randomized the stem of the hairpin there were much more stringent sequence requirements in order to get efficient switching. These data indicated that canonical 4 base pairing and the location of the base pairs on the hairpin stem is crucial for the timely formation of the terminator.

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