Undergraduate Honors Thesis


Aptamer domain stability is prioritized over expression platform stability in a simplified variant of the Bacillus subtilis pbuE adenine-responsive riboswitch Public Deposited

  • Tight regulation of transcription is essential for maintaining life. Transcriptional termination is usually accomplished through Rho-dependent termination or intrinsic termination, the latter of which is often regulated through the use of riboswitches. Riboswitches, which are found in the 5’ untranslated region of mostly bacterial mRNAs, are composed of two domains: the aptamer domain, which is able to bind a ligand, and the expression platform, which controls gene regulation by switching between two alternative structures. Those riboswitches involved in transcriptional control function by switching between a structure with an intrinsic terminator in the expression platform, thereby inhibiting transcription, and a structure that prevents intrinsic terminator formation, allowing transcription to proceed. The conformational switch is triggered when the aptamer domain of the riboswitch binds a small molecule metabolite that is normally associated with the downstream gene, therefore creating a regulatory feedback loop. Many details of the mechanism of this conformational switch remain unknown; therefore, studies of the structural elements required for a functional riboswitch are necessary in order to develop synthetic riboswitches are biosensors and regulatory devices. Characterization of the expression platform is an especially important goal because most previous work has focused on the aptamer domain. For this reason, a series of variant libraries, each with 5-6 different randomized nucleotides in the expression platform, was designed in a simplified variant of the Bacillus subtilis pbuE adenine-responsive riboswitch. The variants within these libraries were 4 inserted ahead of a GFP reporter construct in vivo in order to determine the effect of sequence changes on transcriptional regulation in response to ligand availability. To investigate the combined effects of the aptamer domain and expression platform on transcriptional regulation, the P1-Exchange Library was designed. It contains six randomized nucleotides: two randomized nucleotides in the aptamer domain, two in the expression platform, and two that participate in both the aptamer domain and the expression platform. Variants with low levels of inappropriate downstream gene expression and a high fold-induction of appropriate downstream gene expression were selected for because these characteristics will be necessary in synthetic riboswitches. Functional variants of the P1-Exchange Library were found to have a stronger preference for stable base stacking in the aptamer domain than the expression platform.

Date Awarded
  • 2023-03-15
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Last Modified
  • 2023-04-18
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