. A widespread mechanism of gene regulation in bacteria is by a group of noncoding RNA elements 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 instructs the expression machinery (generally 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 numerous 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. Of equal importance, riboswitches are 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) mapping sequence and structural features of the expression platform beyond the structural switch crucial for efficient ligand-dependent regulatory activity, (2) understanding how structural ?modules? that mediate higher-order tertiary structure contribute to rapid and efficient folding of the aptamer domain, and (3) investigating the plasticity of aptamer domains through their ability to recognize different ligands. 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. Significantly, this proposal adopts a ?function-first? research strategy, as opposed to the ?structure-first? approach that dominates current research into riboswitches to make a stronger connection between RNA structure and function. A deeper knowledge of how RNA specifically interacts with small molecules that affects its structure and activity will contribute to ongoing efforts to develop a new generation of therapeutics that target non-protein coding RNAs in bacteria and eukaryotes.

Public Health Relevance

(Relevance to Public Health). Riboswitches control expression of genes essential for survival or virulence in medically important pathogens such as S. aureus, P. aeruginosa and C. difficile. This research seeks to develop an atomic-level and mechanistic understanding of how riboswitch RNAs regulate gene expression through their ability to directly bind small molecule metabolites. These studies will advance 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM073850-14
Application #
9764705
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Sakalian, Michael
Project Start
2005-04-01
Project End
2023-08-31
Budget Start
2019-09-01
Budget End
2020-08-31
Support Year
14
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Colorado at Boulder
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80303
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Polaski, Jacob T; Kletzien, Otto A; Drogalis, Lea K et al. (2018) A functional genetic screen reveals sequence preferences within a key tertiary interaction in cobalamin riboswitches required for ligand selectivity. Nucleic Acids Res 46:9094-9105
Miao, Zhichao; Adamiak, Ryszard W; Antczak, Maciej et al. (2017) RNA-Puzzles Round III: 3D RNA structure prediction of five riboswitches and one ribozyme. RNA 23:655-672
Polaski, Jacob T; Webster, Samantha M; Johnson Jr, James E et al. (2017) Cobalamin riboswitches exhibit a broad range of ability to discriminate between methylcobalamin and adenosylcobalamin. J Biol Chem 292:11650-11658
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Polaski, Jacob T; Holmstrom, Erik D; Nesbitt, David J et al. (2016) Mechanistic Insights into Cofactor-Dependent Coupling of RNA Folding and mRNA Transcription/Translation by a Cobalamin Riboswitch. Cell Rep 15:1100-1110
Marcano-Velázquez, Joan G; Batey, Robert T (2015) Structure-guided mutational analysis of gene regulation by the Bacillus subtilis pbuE adenine-responsive riboswitch in a cellular context. J Biol Chem 290:4464-75
Trausch, Jeremiah J; Marcano-Velázquez, Joan G; Matyjasik, Michal M et al. (2015) Metal Ion-Mediated Nucleobase Recognition by the ZTP Riboswitch. Chem Biol 22:829-37
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

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