Abstract
Dynamics are central to the function of biomolecules. In the field of RNA, riboswitches are a prime example where regulatory function is encoded by structural transitions. In this work, we used single-molecule fluorescence spectroscopy together with molecular simulations to probe such onformational dynamics. The first part of this thesis reviews established and novel labeling approaches to site-specifically tag nucleic acids with fluorescent markers for Förster resonance energy transfer (FRET) applications. We characterize bioconjugated dyes in terms of their photophysics and introduce computational tools that help in selecting informative distance coordinates.
Biologically active RNA molecules are composed of recurrent, well conserved modules connecting secondary and tertiary structure. Their systematic annotation over several decades led to the notion that RNA folding can be understood by the thermodynamics and kinetics of the constituting building blocks. Here, we chose a long-range tertiary contact reaching from the core of a group II intron to its anking 50-exon. The structure of the isolated contact was previously solved by NMR and allowed us to link chemical features of the ribose backbone and metal ion coordination with dissociation rates measured by single-molecule FRET. We speculate that kinetic heterogeneity in exon recognition has important implications on ribozyme catalysis.
Finally, we turn to a coenzyme B12 riboswitch whose mechanism has remained elusive owing to its structural complexity. Based on the consensus sequence and fragments of other cobalamin riboswitches we built a homology model of the E. coli btuB RNA and probed its dynamics by single-molecule FRET. We found a Mg2+ dependent conformational equilibrium which is thought to coordinate folding of the metabolite binding aptamer with the peripheral expression platform.
Steffen, Fabio D