Towards Investigation of RNA Catalysis and Compartmentalization

Restricted (Penn State Only)
Author:
Frankel, Erica Ann
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
September 12, 2017
Committee Members:
  • Philip C. Bevilacqua, Dissertation Advisor
  • Philip C. Bevilacqua, Committee Chair
  • Scott A Showalter, Committee Member
  • Amie Kathleen Boal, Committee Member
  • James Kasting, Outside Member
  • Christine Dolan Keating, Dissertation Advisor
  • Christine Dolan Keating, Committee Chair
Keywords:
  • RNA
  • Catalysis
  • Compartmentalization
  • Cooperativity
  • Origins of Life
Abstract:
The assembly of molecules and their subsequent reactions that led to the origins of life on Earth nearly four billion years ago remains one of the most elusive mysteries in modern science. One of the first steps scientists look along this timeline is how potentially important molecules, such as early forms of peptides, lipids, and nucleic acids, could have formed. In some cases, it seems plausible that these molecules could have been created on Earth through a sequence of reactions with simple precursors, including methane and cyanide, catalyzed by an electric shock. In other cases, it is proposed that meteors that hit the Earth’s surface brought a menagerie of early molecules that could have acted as precursors for the biomolecules that are pertinent in modern biology today. A second step along this timeline that scientists are particularly interested in is how these newly formed molecules could have been concentrated to form some of the first protocells. Environmental conditions such as warm little ponds, which circuitously evaporate and refill, and porous rock, which would attract various charged molecules to their surface, are examples where potentially important molecules could concentrate, interact, and form the first non-membranous compartments. These highly concentrated environments could have facilitated some of the first autocatalyzed reactions, such as ligation, polymerization, and cleavage. RNA was thought to play a key role in some of these initial pertinent reactions, for its dual functionality to autocatalyze self-cleavage/self-ligation reactions and store genetic information. A third step represents a progression from the second, through the cultivation of more advanced self-catalytic systems. One of the biggest challenges in the origins of life is the ability for a system to self-replicate. Under most RNA systems, RNA replication uses a template strand to build a complementary strand off of, one nucleotide at a time. One limitation for early replication methods is strand dissociation of the product-template complex in order to promote turnover of the template. Again, the earliest primordial systems could have used environmental changes such as temperature and pH to denature template-product complexed to allow for turnover to occur. It is important to note that these steps are relatively arbitrary and most likely did not happen sequentially. It is more than possible that many of these events could have occurred at the same time. Moreover, there are many other steps not included herein, and it is almost certainly the case that the number of steps to reach the earliest organisms may be countless. This thesis, however, lies right at the cusp of steps two and three, as it focuses on the compartmentalization and catalysis of RNA molecules. One key obstacle to forming longer polymers of progenitor molecules would be to concentrate precursor molecules into compartments. In Chapter Two, I characterize the compartmentalization of nucleotides in the presence of a long polyamine. The combination of these two charged molecules spontaneously formed liquid phase droplets in solution, which are referred to as complex coacervates. I find that these coacervate droplets could strongly partition these nucleotides and metal ions, reaching concentrations in the molar regime. Having non-membranous protocells, rich in these types of molecules would may have been important for the generation of primitive polymers. It seems unlikely that earliest forms of RNA would have catalyzed self-cleavage reactions using only one mechanistic pathway. In Chapter Three, we investigate one small RNA enzyme, the hammerhead ribozyme, which uses different methods to activate the general base for catalysis, depending on the environmental conditions. It explicitly reveals RNA’s adaptability to efficiently auto-catalyze reactions no matter the conditions, by means of perturbing nucleobase pKa’s. This theme is carried out into Chapter Four, which broadens the scope of pKa shifting into a platform that can be utilized by RNA and protein scientists. Often pKa shifting can significantly increase catalytic efficiency, yet is seldom observed in experiments. This chapter uses thermodynamics to probe pH dependent interactions that would otherwise be undetected by traditional experimental methods. The thesis merges into the third step in Chapter Five with the evidence of multiple turnover in the hammerhead ribozyme. Under these scenarios, we find that the turnover of the enzyme strand can be easily facilitated by simple changes to environmental conditions. In solutions of high pH and high temperature, the ribozyme can readily turnover nearly 200 times without loss of integrity or degradation. This is quite insightful for drawing some conclusions on how Earth’s environment could have accelerated the generation of more advanced catalytic molecules. In summary, my thesis supports some of the hypotheses leading to the earliest forms of life on Earth. Moreover, some of the phenomena described herein, including the formation of RNA rich non-membranous compartments and pKa shifting, are all found in extant cells, bridging the gap between the earliest primordial chemistry and modern cellular biology.