To respond to rapidly-changing stresses in their environment, bacterial cells must be able to sense a variety of chemical cues and respond to them by activating the relevant genes. The lysine riboswitch is a short RNA motif, located just upstream of a gene encoding a lysine biosynthesis protein, that suppresses the expression of that gene when sufficient lysine is present in the cell. It acts by binding a lysine monomer in a region called the aptamer, which in turn rearranges an adjacent domain called the expression platform, sequestering the ‘start' sequence of the gene and preventing it from being transcribed. In this thesis, the lysine riboswitch's ligand-binding transition is studied using single-molecule fluorescence microscopy, optical tweezers, and a hybrid optical force/fluorescence technique. Förster Resonance Energy Transfer (FRET) is used with a fluorescently-labeled aptamer to show that it has a previously-undescribed, partially-folded structural state with enhanced ligand affinity compared to the unfolded structure. The Mg²⁺ dependence of the transition between these states is shown to resolve existing debates in the literature about the sensitivity of the riboswitch. The kinetics of the folding transition are explored using FRET, optical force, and hybrid ‘Fleezers' to map the free energy landscape of ligand binding and show that the ligand itself promotes transitions into the aptamer's folded state, a so-called ‘induced fit' mechanism rare among riboswitches. Finally, high-resolution optical tweezers are used to explore the link between the aptamer's secondary structure (the sequence of paired nucleotides) and its tertiary structure (three-dimensional folding) to illuminate the role of ligand binding in gene regulation, which depends on the equilibrium between competing secondary structures. Hybrid biophysical techniques like optical force/fluorescence microscopy are shown to be indispensable for addressing all the states in the reaction pathways of complex biomolecules like riboswitches and for discriminating between multiple levels of structure formation and interaction with the environment. Not only do the results presented here shed light on the RNA folding problem, particularly the role of tertiary structure in determining the minimum-energy configuration of an RNA sequence, but they could have implications for biomedical research, as the lysine riboswitch has already been shown to be a potential target for next-generation antibiotics.

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<div> <div> <div> <p>The cosmos of noncoding RNAs (ncRNAs) has been thriving in recent years; so much so that researchers are discovering them much faster than they can uncover their functions. The subset of these RNAs that have been characterized have been noted to perform and regulate a plethora of remarkably diverse and essential biological functions. This diversity in function is accompanied by a large array of dynamic and elegantly folded 3-dimensional structures. In this collection of work, we will journey through the discovery of the first catalytic noncoding RNAs (ribozymes), explore a new method for identifying uniquely structured ribozymes, and detail the design of a technique to select for highly structured RNAs with a high affinity for an RNA binding partner. Although these topics vary widely within the field of RNA, this work strives to showcase the integral relationship between intricate macromolecular structures with their chemical and cellular functions. </p> </div> </div> </div>

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Non-coding RNAs are involved in the regulation of most major cellular process in Escherichia coli. With current technologies, many of these molecules have been identified; however, the full scope of their regulatory interactions is still unknown. None of the techniques currently in use employ the regulatory effect of the RNAs, which is the major unifying attribute of these molecules, for their identification. This thesis presents projects involving the design of a dual-reporter plasmid and screening method in the discovery and characterization of RNA regulatory interactions The first project details the engineering of the dual reporter plasmid. This vector utilizes one fluorescent protein to detect regulatory events and a second to normalize for off-target effects. The second project utilizes this tool in the discovery and characterization of novel regulatory responses. This is accomplished by screening a library of intergenic regions for regulatory responses against a collection of metabolite. Interesting interactions involving nitrogen abundance, iron and uracil are identified and further examined. Finally, this thesis examines how this technology can be further expanded for the study of RNA regulatory functions. The use of the screening method for the detection of regulatory events caused by alternative minimal media composition and the potential for the dual reporter plasmid to aid in the study of riboswitches are investigated. / Thesis / Doctor of Science (PhD)

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Non-protein-coding RNAs play critical regulatory roles in cellular life. Many ncRNAs fold into specific structures in order to perform their biological functions. Some of the RNAs, such as riboswitches, can even fold into alternative structural conformations in order to participate in different biological processes. In addition, these RNAs can transit dynamically between different functional structures along folding pathways on their energy landscapes. These alternative functional structures are usually energetically favored and are stable in their local energy landscapes. Moreover, conformational transitions between any pair of alternate structures usually involve high energy barriers, such that RNAs can become kinetically trapped by these stable and local optimal structures. We have proposed a suite of computational approaches for analyzing and discovering regulatory RNAs through studying folding pathways, alternative structures and energy landscapes associated with conformational transitions of regulatory RNAs. First, we developed an approach, RNAEAPath, which can predict low-barrier folding pathways between two conformational structures of a single RNA molecule. Using RNAEAPath, we can analyze folding iii pathways between two functional RNA structures, and therefore study the mechanism behind RNA functional transitions from a thermodynamic perspective. Second, we introduced an approach, RNASLOpt, for finding all the stable and local optimal structures on the energy landscape of a single RNA molecule. We can use the generated stable and local optimal structures to represent the RNA energy landscape in a compact manner. In addition, we applied RNASLOpt to several known riboswitches and predicted their alternate functional structures accurately. Third, we integrated a comparative approach with RNASLOpt, and developed RNAConSLOpt, which can find all the consensus stable and local optimal structures that are conserved among a set of homologous regulatory RNAs. We can use RNAConSLOpt to predict alternate functional structures for regulatory RNA families. Finally, we have proposed a pipeline making use of RNAConSLOpt to computationally discover novel riboswitches in bacterial genomes. An application of the proposed pipeline to a set of bacteria in Bacillus genus results in the re-discovery of many known riboswitches, and the detection of several novel putative riboswitch elements.

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