UVM Theses and Dissertations
Format:
Print
Author:
Brooks-Greco, Krista
Dept./Program:
Microbiology and Molecular Genetics
Year:
2011
Degree:
PhD
Abstract:
In the last thirty years, the functional role of RNA in the cell has been reevaluated and significantly revised. Like DNA, RNA transmits genetic information, yet RNA functions beyond the capabilities of DNA. In addition to serving as an intermediary, conveying genetic information from DNA to protein, RNA has also been shown to function in regulating gene expression and also to possess enzymatic activity. To modulate gene expression and perform enzymatic reactions independent of proteins, RNA must utilize ions and metabolites within its environment. Riboswitches are RNA structural elements that regulate gene expression through conformational rearrangements upon binding a cognate ligand. These conformational changes result in transcription termination via formation of hairpin structures or can inhibit translation by sequestering the ribosome-binding site. The glmS ribozyme differs from other riboswitches in that it possesses enzymatic activity and is able to preform its tertiary structure, including its metabolite binding pocket, in the absence of its cognate ligand, glucosamine 6-phosphate (GlcN6P).
RNA must undergo complex folding to achieve a specific tertiary structure, analogous to that of a protein, when acting as a ribozyme. To improve our understanding of the glmS ribozyme, it is important to dissect the folding and catalytic pathways to elucidate the role that cations play in global folding, ligand binding, and catalysis and also to better understand the role of GlcN6P in folding and catalysis. Dissection ofthe catalytic pathway revealed that self-cleavage reactions initiated with simultaneous addition of Mg² and GlcN6P are slow (~3 min⁻¹) compared to reactions initiated by addition of GlcN6P to glmS RNA that has been prefolded in Mg²⁺-containing buffer (~72 min⁻¹) . Our findings indicate that some level of Mg²⁺-dependent folding is rate-limiting for catalysis. Time-resolved hydroxyl-radical footprinting was employed to determine if global tertiary structure formation is the rate-limiting step. These experiments provided evidence for fast and largely concerted folding of the global tertiary structure (>13min⁻¹) . This indicates that the rate-limiting step that we have identified either is a slow folding step between the fast initial folding and ligand binding events or represents the rate of escape from a nativelike folding trap.
Additionally, metal ions play important roles in the formation of tertiary structure and catalytic function of RNA molecules. The glmS ribozyme has previously been shown to lack strong cation specificity, as defined by assays that measure the rate-limiting step of the cleavage reaction pathway. Our previous data demonstrated that prefolding of the ribozyme in Mg²-containing buffers effectively isolates the rapid ligand binding and catalytic events (kobs> 60 min⁻¹) from this rate-limiting folding step (kobs<4 min⁻¹) . Here we provide evidence that molar concentrations ofmonovalent cations are also capable of inducing the formation of the native GlcN6P binding structure, but are unable to promote ligand binding and catalysis rates greater than 4 min⁻¹. A potential role for divalent cations, for which there is crystallographic evidence, is coordination ofthe phosphate moiety of GlcN6P in the ligand binding pocket. In support ofthis hypothesis, our data show that a non-phosphorylated analog of GlcN6P, glucosamine, is unable to promote rapid ligand binding and catalysis in the presence of divalent cations.
RNA must undergo complex folding to achieve a specific tertiary structure, analogous to that of a protein, when acting as a ribozyme. To improve our understanding of the glmS ribozyme, it is important to dissect the folding and catalytic pathways to elucidate the role that cations play in global folding, ligand binding, and catalysis and also to better understand the role of GlcN6P in folding and catalysis. Dissection ofthe catalytic pathway revealed that self-cleavage reactions initiated with simultaneous addition of Mg² and GlcN6P are slow (~3 min⁻¹) compared to reactions initiated by addition of GlcN6P to glmS RNA that has been prefolded in Mg²⁺-containing buffer (~72 min⁻¹) . Our findings indicate that some level of Mg²⁺-dependent folding is rate-limiting for catalysis. Time-resolved hydroxyl-radical footprinting was employed to determine if global tertiary structure formation is the rate-limiting step. These experiments provided evidence for fast and largely concerted folding of the global tertiary structure (>13min⁻¹) . This indicates that the rate-limiting step that we have identified either is a slow folding step between the fast initial folding and ligand binding events or represents the rate of escape from a nativelike folding trap.
Additionally, metal ions play important roles in the formation of tertiary structure and catalytic function of RNA molecules. The glmS ribozyme has previously been shown to lack strong cation specificity, as defined by assays that measure the rate-limiting step of the cleavage reaction pathway. Our previous data demonstrated that prefolding of the ribozyme in Mg²-containing buffers effectively isolates the rapid ligand binding and catalytic events (kobs> 60 min⁻¹) from this rate-limiting folding step (kobs<4 min⁻¹) . Here we provide evidence that molar concentrations ofmonovalent cations are also capable of inducing the formation of the native GlcN6P binding structure, but are unable to promote ligand binding and catalysis rates greater than 4 min⁻¹. A potential role for divalent cations, for which there is crystallographic evidence, is coordination ofthe phosphate moiety of GlcN6P in the ligand binding pocket. In support ofthis hypothesis, our data show that a non-phosphorylated analog of GlcN6P, glucosamine, is unable to promote rapid ligand binding and catalysis in the presence of divalent cations.