Riboswitches are a class of non-protein-coding (nc) RNA elements involved in the regulation of key metabolic pathways in bacteria through small molecule binding. Several of these pathways, such as preQ1 biosynthesis, are unique to eubacteria and represent novel antimicrobial targets to combat human pathogens. To better understand this unique mechanism of gene regulation, we determined ligand-bound and ligand-free crystal structures of a class 1 preQ1 riboswitch during the prior 2-year ARRA funding period. This work provided the first analysis of a preQ1 riboswitch that controls translation, and represents the only structural analysis of a translational riboswitch in the ligand-free (apo) stat. The results provide a tantalizing glimpse of how a cellular metabolite can govern the interaction between mRNA and the ribosome. A comparison of the ligand-bound and ligand-free structures suggested that the conformation of the preQ1-binding pocket is dictated by ligand binding, and that this interaction controls access to a spatially distant ribosome-binding site (RBS).
In Aim #1 of this proposal, we will expand on prior work to evaluate whether a phylogenetically unrelated preQ1 class 2 riboswitch employs the RBS-sequestration mechanism, thereby testing the universality of this gene regulation strategy.
In Aim #2, we will examine the interaction between preQ1 riboswitches and the translation-initiation complex. The recognition of mRNA by the ribosome is a fundamental process, yet no investigation has probed the influence of ribosomes on the riboswitch folding landscape. We have assembled a team of single molecule (sm)FRET and ribosome experts for this aim (Nils Walter, University of Michigan and Dmitri Ermolenko, University of Rochester), and the results should provide a meaningful description of the ligand levels and riboswitch sequences required for translational regulation.
Aim #3 of this proposal is based on our prior analysis of the apo preQ1 riboswitch. Obtaining the apo crystal structure was unexpected since independent studies on transcriptional preQ1 riboswitches showed strict, ligand-dependent folding. For this reason, we used small angle X-ray scattering to demonstrate that the translational preQ1 riboswitch is as compact in solution as the ligand-bound state. However, the apo crystal structure could not adequately recapitulate the apo riboswitch scattering data, suggesting the ligand-free ensemble in solution is complex.
In Aim #3, we will analyze the solution ensembles of preQ1 riboswitches by small angle scattering and in-line probing. This work will require novel computational approaches that will be conducted in collaboration with David Mathews (University of Rochester). These experiments will provide insight into the 'prefoldedness'of the apo state and its receptiveness to ligand binding, as embodied by the principle of conformational selection. The overall results of the proposed investigation will expand our understanding of this unique mechanism of gene regulation for a novel metabolic pathway in bacteria. In the long term, this work can be exploited for the development of novel antimicrobials that have little or no effect on host pathways.

Public Health Relevance

Riboswitches are a widespread class of gene regulatory element that control as many as 4% of genes in some bacteria. In this proposal, we will investigate how conformational changes in a riboswitch that binds the preQ1 metabolite lead to attenuation of protein synthesis. This work will provide broad insight into the mechanism of action of translational riboswitches, which are present in several pathogens that represent emergent public health threats due to drug resistance.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function C Study Section (MSFC)
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Preusch, Peter C
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University of Rochester
Schools of Dentistry
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Heldenbrand, Hugh; Janowski, Pawel A; Giamba?u, George et al. (2014) Evidence for the role of active site residues in the hairpin ribozyme from molecular simulations along the reaction path. J Am Chem Soc 136:7789-92
Liberman, Joseph A; Salim, Mohammad; Krucinska, Jolanta et al. (2013) Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold. Nat Chem Biol 9:353-5
Liberman, Joseph A; Wedekind, Joseph E (2012) Riboswitch structure in the ligand-free state. Wiley Interdiscip Rev RNA 3:369-84
Lippa, Geoffrey M; Liberman, Joseph A; Jenkins, Jermaine L et al. (2012) Crystallographic analysis of small ribozymes and riboswitches. Methods Mol Biol 848:159-84
Jenkins, Jermaine L; Krucinska, Jolanta; McCarty, Reid M et al. (2011) Comparison of a preQ1 riboswitch aptamer in metabolite-bound and free states with implications for gene regulation. J Biol Chem 286:24626-37
Wedekind, Joseph E (2011) Metal ion binding and function in natural and artificial small RNA enzymes from a structural perspective. Met Ions Life Sci 9:299-345
Liberman, Joseph A; Wedekind, Joseph E (2011) Base ionization and ligand binding: how small ribozymes and riboswitches gain a foothold in a protein world. Curr Opin Struct Biol 21:327-34
Wedekind, Joseph E (2010) The apo riboswitch as a molecular hydra. Structure 18:757-8
Spitale, Robert C; Torelli, Andrew T; Krucinska, Jolanta et al. (2009) The structural basis for recognition of the PreQ0 metabolite by an unusually small riboswitch aptamer domain. J Biol Chem 284:11012-6
Guo, Man; Spitale, Robert C; Volpini, Rosaria et al. (2009) Direct Raman measurement of an elevated base pKa in the active site of a small ribozyme in a precatalytic conformation. J Am Chem Soc 131:12908-9

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