Single-molecule FRET investigations of riboswitch-mediated translational control

The goal of the proposed research is to dissect the mechanism by which cellular metabolites transduce signal through defined RNA elements present in specific messenger RNA (mRNA) species to control gene expression. mRNA elements of this kind are collectively referred to as riboswitch domains and operate in bacteria by up- or down-regulating the processes of transcription and/or translation catalyzed by RNA polymerase and the ribosome, respectively. Genome sequencing efforts suggest that riboswitches are ubiquitous in bacterial species, but absent in higher organisms. Recent structural studies have revealed that riboswitches respond to the binding of small- molecule metabolites by adopting one or more structured domains to either mask or expose the nucleic acid template to regulate its interaction with the corresponding gene expression machinery. However, the physical relationship between metabolite-induced riboswitch folding and an mRNA`s ability to interact with the gene expression machinery has yet to be explored. To fill this knowledge gap we will use a combination of physical and organic chemistry initiatives to explore the mechanism of mRNA-mediated translational control at the chemical, biophysical and single-molecule level. In so doing, we will directly assess the correlation between conformational changes in riboswitch structure, ligand binding kinetics and the rate and efficiency of translation initiation processes. Consequently, we will determine at which point riboswitch-mediated regulation occurs during the translation initiation process and what role, if any, is played by the translation machinery. Specific focus will be given to the SAM-II and purine riboswitches, whose switch domains have been unambiguously identified and whose ligand-bound structures have been solved. This work leverages key technologies and imaging strategies developed in the labs of both Principal Investigators through the course of their independent and collaborative efforts that enable the site-specific tagging of the key players in riboswitch-mediated translational control. The anticipated outcomes of this research include fundamentally new insights into the role of dynamics in riboswitch- mediated regulation of protein synthesis that will significantly advance our understanding of this novel means of gene expression control. Intellectual Merit: Although riboswitches are positive or negative regulators of gene expression operating at the level of both transcription and translation in many bacterial organisms, little is presently known about the physical "switching" mechanism itself and how this dynamic process is temporally linked to the process of translation initiation and metabolite sensing. In part, this shortcoming is due to the paucity of biophysical tools capable of monitoring riboswitch dynamics and the process of translation initiation simultaneously. Thus, in addition to providing insights into the principles of riboswitch mediated gene expression control employed by prokaryotic organisms, the proposed research is anticipated to shed important new light on basic aspects of the translation initiation mechanism that more broadly serve as key points of regulatory intervention. Such insights may ultimately reveal how bacterial species can be engineered to possess new, orthogonal means of gene expression control and how riboswitches in pathogenic organisms may be leveraged for therapeutic intervention.

The scientific goal of the project was to shed light on the folding and ligand binding dynamics of riboswitches in the context of ribosomal translation. How riboswitch folding enters distinct pathways upon binding to its dedicated ligand remains elusive despite intensive investigations in recent years. When the project started in 2013, little was known about intrinsic riboswitch folding dynamics and the dynamic process of ligand-RNA recognition that are suspected to represent key determinants at the molecular level for making up the genetic decision. The obvious significance of riboswitches as regulatory units and as potential drug targets, prompted us to put strong efforts into the development of efficient synthetic routes to multiple labeled RNAs that are required for single-molecule fluorescence-resonance-energy-transfer (smFRET) studies to explore their dynamic functional mechanism, the latter performed in close collaboration with Scott Blanchards laboratory (Weill Cornell Medicine). To achieve this goal, several hurdles had to overcome: First, we developed new methods for multi-labeling of RNA. Second, based on these labeling methods, we conducted smFRET investigations in Scott Blanchards laboratory and revealed how RNA long-range secondary structure interactions affect ligand binding and folding of a riboswitch aptamer (TPP riboswitch). Third, we expanded these studies towards another riboswitch (preQ1 riboswitch) in order to reveal how individual RNA secondary structure elements affect ligand binding and folding. Fourth, we complemented smFRET with steady-stated fluorescence and NMR spectroscopic approaches to obtain a comprehensive understanding of riboswitchligand recognition and folding (utilizing the preQ1 systems). Our attempts to study riboswitch mediated translation initiation turned out to be technically complex and warrent further indepth investigations in the future. Furthermore, during the course of the project, four novel ribozyme classes were discovered by Ronald Breakers group. The corresponding RNA secondary models resembled riboswitch motives; we therefore started to extend our studies to these RNAs with the aim to bridge riboswitch folding/regulation to ribozyme folding/self-cleavage. First results on the organization and dynamic behavior of these folds were achieved and published.