A widely used mechanism of gene regulation in bacteria is a noncoding RNA element called a riboswitch. These are cis-acting elements found in the leader sequence of mRNAs and regulate gene expression through their ability to directly bind a specific effector molecule to a highly structured aptamer domain. Effector binding to the aptamer domain is communicated to a downstream secondary structural switch in the expression platform that interfaces with the expression machinery (either RNA polymerase or the ribosome). In a broad spectrum of bacteria, particularly Firmicutes and Fusobacteria, central metabolic pathways including purine, amino acid, and cofactor biosynthesis and transport are regulated by riboswitches. Furthermore, genes essential for survival or virulence are under riboswitch control in a number of medically important pathogens including Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium difficile, and Mycobacterium tuberculosis making them of great interest as novel targets for antimicrobial therapeutics. In addition, riboswitches serve as powerful model systems for understanding various aspects of RNA biology including structure, folding and mechanisms of regulatory activity along with developing tools and methodologies for designing small molecules that target other RNAs of medical interest. Towards the long-term goal of developing a molecular understanding of how RNA interacts with small molecules and the mechanisms it uses to regulate gene expression, we are using purine- and cobalamin- binding riboswitches as model systems. This proposal details a set of interconnected specific aims that addresses fundamental questions related to these research goals: (1) what is the global structure of purine- sensing riboswitches, (2) why do purines inefficiently regulate the THF riboswitch, and (3) how do cobalamin riboswitches functionally couple effector binding to gene regulation? To address these questions, a combination of structural (x-ray crystallography) biophysical (calorimetry and stopped-flow kinetics), biochemical (chemical footprinting), genetic and molecular biological (in vivo and in vitro activity assays) and bioinformatics/computational approaches will be combined to study the structure-regulatory activity linkage in riboswitches. A deeper knowledge of how RNA specifically interacts with small molecules and affect its structure and activity will help pav the way for a new generation of therapeutics that target non-protein coding RNAs in bacteria and eukaryotes.

Public Health Relevance

Riboswitches are one of the many forms of RNA-based gene regulation in bacteria and eukarya. In some medically important pathogens such as S. aureus, P. aeruginosa, and C. difficile these regulatory elements control expression of genes essential for survival or virulence, suggesting that they could be important new targets for antimicrobial agents. The proposed research seeks to develop an atomic-level understanding of how these RNAs regulate gene expression through their ability to directly bind small molecules. These studies will both further our understanding into basic mechanisms of bacterial physiology and yield new insights into how to exploit RNA as a target of small-molecule therapeutics through structure-based drug design.

National Institute of Health (NIH)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Preusch, Peter
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Colorado at Boulder
Schools of Arts and Sciences
United States
Zip Code
Batey, Robert T (2014) Advances in methods for native expression and purification of RNA for structural studies. Curr Opin Struct Biol 26:1-8
Trausch, Jeremiah J; Batey, Robert T (2014) A disconnect between high-affinity binding and efficient regulation by antifolates and purines in the tetrahydrofolate riboswitch. Chem Biol 21:205-16
Holmstrom, Erik D; Polaski, Jacob T; Batey, Robert T et al. (2014) Single-molecule conformational dynamics of a biologically functional hydroxocobalamin riboswitch. J Am Chem Soc 136:16832-43
Porter, Ely B; Marcano-Velázquez, Joan G; Batey, Robert T (2014) The purine riboswitch as a model system for exploring RNA biology and chemistry. Biochim Biophys Acta 1839:919-930
Ceres, Pablo; Trausch, Jeremiah J; Batey, Robert T (2013) Engineering modular 'ON' RNA switches using biological components. Nucleic Acids Res 41:10449-61
Stoddard, Colby D; Widmann, Jeremy; Trausch, Jeremiah J et al. (2013) Nucleotides adjacent to the ligand-binding pocket are linked to activity tuning in the purine riboswitch. J Mol Biol 425:1596-611
Ceres, Pablo; Garst, Andrew D; Marcano-Velazquez, Joan G et al. (2013) Modularity of select riboswitch expression platforms enables facile engineering of novel genetic regulatory devices. ACS Synth Biol 2:463-72
Garst, Andrew D; Edwards, Andrea L; Batey, Robert T (2011) Riboswitches: structures and mechanisms. Cold Spring Harb Perspect Biol 3:
Trausch, Jeremiah J; Ceres, Pablo; Reyes, Francis E et al. (2011) The structure of a tetrahydrofolate-sensing riboswitch reveals two ligand binding sites in a single aptamer. Structure 19:1413-23
Daldrop, Peter; Reyes, Francis E; Robinson, David A et al. (2011) Novel ligands for a purine riboswitch discovered by RNA-ligand docking. Chem Biol 18:324-35

Showing the most recent 10 out of 19 publications