TOWARDS THE INVESTIGATION OF THERMALLY STIMULATED MULTIPLE TURNOVER IN THE HAMMERHEAD RIBOZYME

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Author:
Shelton, Kimberly Kathleen
Area of Honors:
Chemistry
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Philip Bevilacqua , Thesis Supervisor
  • Raymond Funk , Honors Advisor
  • Andrew Zydney, Honors Advisor
Keywords:
  • ribozyme
  • RNA World Hypothesis
  • hammerhead ribozyme
  • temperature dependence
  • thermocycling
Abstract:
The RNA world hypothesis states that, prior to the generation of protein enzymes and DNA, RNA would have been the essential molecule to fill both roles as a catalyst and as the carrier of genetic information. This hypothesis supports the idea that RNA catalyzed RNA replication may have contributed to the basis for the origins of early life. However, this reaction is limited by many factors, one of which includes strand dissociation of product for the multiple turnover of the enzyme. Therefore, it is important to elucidate ways in which strand dissociation could have been mediated without the need for modern cellular machinery. To lend support to this hypothesis, I have been investigating the single and multiple turnover reactions of the hammerhead ribozyme as a function of temperature at neutral pH as a way to mimic thermal vent environments and facilitate strand dissociation. Studies conducted on a wild type hammerhead construct found that strand dissociation under the multiple turnover regime occurred at low and high temperatures, so reactions could not be decoupled between the reaction step and strand dissociation step. A new thermophilic construct was thus generated with stronger base pairing to allow for experiments to be completed at higher temperatures to observe more distinct dissociation steps within the multiple turnover reactions. Results for this construct suggested a temperature dependency, indicating that strand dissociation occurs more easily at higher temperatures due to the inherent increase in energy needed to break hydrogen bonds between strong base pairs. Thermocycling experiments of the thermophilic construct demonstrated that substrate cleavage occurred at intermediate temperatures while product dissociation occurred at high temperatures, supporting the theory that under the right conditions, successive temperature change in deep sea hydrothermal vents could have facilitated the buildup of genetic material necessary for the origins of life here on earth.