Structural and kinetic studies of tertiary folding of an unmodified transfer RNA

The process by which linear sequences of amino acids and nucleotides fold into the complex three-dimensional structures observed in proteins and RNA has intrigued researchers for many years. A solution to the folding problem for both proteins and nucleic acids requires an understanding of the structure and energetics of each species along the folding pathway, including the transition states associated with conversion between different conformational states. This thesis presents experiments designed to study the structure and transition state of tertiary folding of unmodified yeast tRNA{dollar}rmsp{lcub}Phe{rcub}{dollar} using engineered disulfide bonds.; Chapter 2 describes the design, synthesis, and structural characterization of three unmodified yeast tRNA{dollar}rmsp{lcub}Phe{rcub}{dollar} analogs possessing a disulfide cross-link bridging units of secondary or tertiary structure. These experiments demonstrate that disulfide cross-links can be incorporated into complex regions of tertiary structure without distorting the folded structure. To measure the activation parameters of tertiary unfolding of these constructs requires both a method that can monitor fast folding and determination of solution conditions that support secondary and tertiary structure formation. Chapter 3 discusses the development of a kinetic method that monitors tertiary unfolding transitions in tRNA on millisecond timescales exploiting the Mg{dollar}sp{lcub}2+{rcub}{dollar} dependence of tRNA folding. Chapter 4 describes the structural, thermodynamic, and kinetic properties of unmodified yeast tRNA{dollar}rmsp{lcub}Phe{rcub}.{dollar} The results of this work suggest that the tertiary structure of the unmodified yeast tRNA{dollar}rmsp{lcub}Phe{rcub}{dollar} is in a Mg{dollar}sp{lcub}2+{rcub}{dollar}-dependent equilibrium and define conditions to monitor tertiary unfolding.; In chapter 5 the disulfide cross-linked tRNAs are used to examine the transition state of tertiary unfolding of unmodified yeast tRNA{dollar}rmsp{lcub}Phe{rcub}.{dollar} This method uses the disulfide bond as a probe for when the tethered regions interact with respect to the transition state of tertiary unfolding. These experiments suggest that tertiary structure at the interface of the D- and T-loops unfolds prior to or during the rate determining step of tertiary unfolding. Information about the transition state of unfolding is important to predict tertiary structure of an RNA from secondary structure. The disulfide engineering method presented here allows investigation of the structure of the tertiary unfolding transition state, which should be applicable to other RNAs.