Investigating the Folding of the Class I Type II PreQ1 Riboswitch in Cellular Conditions

Open Access
Author:
Luteran, Emily Michelle
Area of Honors:
Chemistry
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Philip C. Bevilacqua, Thesis Supervisor
  • Raymond Lee Funk, Honors Advisor
  • Scott A Showalter, Faculty Reader
  • Xin Zhang, Faculty Reader
Keywords:
  • preQ1
  • riboswitch
  • RNA
  • folding
  • cellular conditions
Abstract:
Historically, RNA is thought to be a messenger molecule that transfers genetic information between DNA and proteins. However, recent studies have identified many necessary functions of RNA beyond acting as a messenger such as RNA enzymes, RNA thermometers, and RNA gene switches. The structures that an RNA adopts often dictate its function, in a similar manner to protein folding. RNA folding has been studied extensively under dilute in vitro solution conditions, yet little is known about RNA folding in vivo. In vitro studies are typically performed in high concentrations of Mg2+ and Na+, which is very different from the cellular environment. The goal of this project is to get a better understanding of riboswitch folding under in vivo-like conditions, through a focus on the smallest riboswitch known, the preQ1 riboswitch. Riboswitches are RNAs that control gene expression by binding to small molecules in cells and changing structure. In particular, the preQ1 riboswitch binds to the preQ1 metabolite in the cell to regulate queuosine synthesis. In vivo-like studies were performed in solution with physiologically low concentrations of Mg2+ and K+, along with 20-40% molecular crowders to mimic the presence of macromolecules in the cell1. The folding of the preQ1 riboswitch was studied by thermal denaturation experiments under in vitro and in vivo-like conditions. Results to date suggest to RNA stabilization with and without preQ1 ligand in the presence of 10-20% crowder (PEG1K and PEG8K), along with an increase in cooperative, two-state folding under these conditions. Further work entails structure mapping the RNA to assign each melting transition to specific segments of the RNA and to examine how the in vivo-like folding of the riboswitch changes as the size of the RNA is changed and, ultimately, limited to the minimum aptamer domain.