toward understanding RNA structure, folding, and function in cells

Open Access
Author:
Strulson, Christopher A
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
January 30, 2014
Committee Members:
  • Philip C. Bevilacqua, Dissertation Advisor
  • Christine Dolan Keating, Dissertation Advisor
  • Squire J Booker, Committee Member
  • Tae Hee Lee, Committee Member
  • Kenneth Charles Keiler, Committee Member
Keywords:
  • RNA
  • ribozyme
  • molecular crowding
  • macromolecular crowding
  • catalysis
  • compartmentalization
  • folding cooperativity
Abstract:
Ever since the discovery of RNA as an integral biomolecule in cellular function, the principles governing RNA folding and function have been studied. These studies have primarily focused on the thermodynamics and kinetics of RNA folding in conditions that are folding-favorable and non-physiological including dilute solution with relatively high ionic strength and Mg2+ ion concentrations. While these studies have imparted invaluable knowledge towards the understanding of RNA biophysics, there is still a great need to characterize RNA folding and function in conditions that are much closer to those of a cell; however, because of the intricacies of the cellular environment, elucidating meaningful biophysical properties from experiments directly in living cells remains difficult. In an effort to capture the effects of this complex environment on RNA folding, in vitro experiments focusing on distinct features of the cellular environment are performed in attempt to learn how these specific properties of the cellular matrix impact RNA. In this thesis, RNA structure, folding, and function are assessed from both a thermodynamic and kinetic prospective under conditions that mimic those of cells. In particular, effects of compartmentalization, macromolecular crowding, and low molecular cosolutes are examined in terms of RNA catalysis and RNA folding using an array of techniques including RNA partitioning, ribozyme kinetics, small angle X-ray scattering (SAXS), structure mapping methods, and RNA folding thermodynamics. One key aspect of cells is their ability to compartmentalize biomolecules; thus, allowing them to control local concentrations of these molecules. In Chapter Two and Three of this thesis, it is revealed that local concentrations of RNA can be controlled using an aqueous two-phase system composed of two high molecular weight polymers. Additionally, increasing the local concentration of an enzyme strand of a two-piece ribozyme within one of the aqueous phase compartments, leads to a significant enhancement in catalysis under single-turnover conditions. This work indicates that compartmentalization could be very important for RNA function in modern cells as well as in RNA regulation and activation in early-earth protocells. Another key factor of the cellular environment is the concentration of free Mg2+, which is only ~0.5 mM and ~2 mM in eukaryotic and prokaryotic cells, respectively. These values are considerably lower than the free Mg2+ (~10 mM) typically used in RNA structure and folding studies in the literature. Additionally, there are high concentrations of molecules (both high- and low-molecular weight) inside cells that can interact with RNA, exclude volume, and alter solvent conditions. These molecules can have large impacts on RNA structure, folding, and function especially when Mg2+ concentrations are physiological. It is revealed in Chapter Four of this thesis that the self-cleavage of a small catalytic RNA, the cytoplasmic polyadenylation element–binding protein 3 HDV-like ribozyme (CPEB3), is stimulated by high- and low-molecular weight molecules under physiological free Mg2+ concentrations. Furthermore, SAXS experiments reveal compactness of the ribozyme under such conditions. These findings suggest RNA structure-function-relationships that uniquely arise from cellular conditions. To link the RNA folding pathway with RNA activity assays in cellular conditions, in Chapter Five, folding cooperativity of RNA was assessed in physiological concentrations of Mg2+, macromolecular crowders, and low molecular weight cosolutes. Through a combination of biophysical characterization and structure mapping techniques, it was found that tRNAPhe folding cooperativity increases in the presence of macromolecular crowding agents under physiological concentrations of Mg2+. Conversely, in physiological concentrations of Mg2+ and low molecular weight cosolutes, tRNAPhe folding cooperativity is either increased or decreased depending on the identity of the cosolute. These experiments demonstrate that cellular conditions increase the steepness of the unfolding transition for RNA and may possibly facilitate native RNA folding despite having low concentrations of free Mg2+. The work presented in this thesis reveals the effects of the cellular environment on RNA structure, folding, and function and further strengthens the biological structure-function relationship that is so vital to RNA behavior.