Genetic Screen of the B. subtilis pbuE Adenine-Responsive Riboswitch Expression Platform Reveals Preferences for Base Pairing in the Nucleator Hairpin-Stem

Altenbern, Ava Lorraine

Undergraduate Honors Thesis

Genetic Screen of the B. subtilis pbuE Adenine-Responsive Riboswitch Expression Platform Reveals Preferences for Base Pairing in the Nucleator Hairpin-Stem Öffentlichkeit Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/s7526f00s

Abstract

Located on the 5’ untranslated regions of bacterial messenger RNA, riboswitches are regulatory structures that are responsible for the modulation of genetic expression through ligand-dependent binding. Inclusive of two components, the riboswitch will probe its environment without the aid of an additional protein or DNA structure to sense and attach a specific metabolite to the region known as the upstream aptamer domain. The downstream expression platform then endures changes in its folding pattern to adopt one of two secondary structures, resulting in either the inhibition or continuation of mRNA production. Due to its smaller size, the B. subtilis pbuE adenine-responsive riboswitch has been the focus of many previous studies that sought to determine how the tertiary structure of the aptamer domain allows for tight binding with high specificity. The expression platform, however, is similarly interesting, as it participates in strand invasion in order to produce a transcription terminating hairpin that is rho independent.

Through the mutagenic cloning of a novel pbuE variant named NH5, the investigation into the reduced nucleator Hairpin-Stem library containing 6 randomized nucleotides revealed a strict preference for genetic base pairing proximal to the L4 loop. Additionally, the data suggests that weak A-U and G-U interactions or even non-canonical coupling between the nucleobases furthest from the polyuridine tract is tolerated if supplemented with three strong Watson-Crick pairs. Paving the way for the creation of additional synthetic riboswitch structures, the robust screening of the P4 region allows for a more thorough understanding of the fundamental requirements that promote the formation of the terminator helix and the subsequent mechanism of strand invasion.

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