Date of Award
Master of Science (MS)
Chemistry & Biochemistry
Robert T. Batey
The hammerhead motif comprises a family of small self-cleaving ribozymes whose involvement in post-transcriptional regulation of gene expression is rapidly becoming more appreciated across biological systems. Structural studies have revealed that the Schistosoma mansoni hammerhead achieves catalytic activity under physiological conditions via the formation of a highly specific tertiary interaction that positions the essential nucleotides to participate in acid-base chemistry. While this interaction forms efficiently in sub-millimolar concentrations of any divalent metal ion, a great deal of metal ion specificity is observed for enzymatic cleavage. Previous experiments have used single-molecule Förster resonance energy transfer (FRET) to interrogate this phenomenon, but have encountered structural dynamics that appear to be artificially coupled to the fluorescence intensity of the dye, raising the concern that the fluorophores are interfering with the intrinsic structural dynamics of the ribozyme. Here, alternative FRET labeling strategies are explored. Fluorophore positions within the interacting stems were tested for enzymatic activity, but modification resulted in diminished catalysis in all cases. The focus was therefore shifted to an orthogonal labeling strategy that tracks the formation of the tertiary interaction indirectly through the coaxial stacking of two non-interacting stems. Magnesium ion-dependent FRET was observed with this construct under single-molecule conditions, albeit with a smaller change in FRET upon conformational reorganization. The conformational states could be distinguished reliably by implementing an improved Hidden Markov modeling algorithm, allowing a side-by-side comparison between this and the original construct using smFRET. Ostensible differences in kinetics and heterogeneity, particularly for the unfolding transition, were observed for the structural dynamics of the two constructs. These results are formally consistent with steric inhibition of the tertiary interaction from the fluorophore, or that the natural structural dynamics are in some other way affected by fluorophore incorporation strategy. Alternatively, the differences could arise from incongruity between respective FRET labeling strategies' ability to distinguish conformational states of the ribozyme.
Swan, Jeffrey Alan, "The Effect of Fluorescent Labeling on Structural Dynamics and Catalysis of the Schistosoma mansoni Hammerhead Riboszyme" (2014). Chemistry & Biochemistry Graduate Theses & Dissertations. 123.