In bacteria, a widely used means of genetic regulation is a non-protein coding RNA element called a riboswitch. These RNA sequences are cis-acting elements found in the 5'-untranslated region (5'-UTR) of mRNAs and regulate gene expression via their ability to directly bind small molecule metabolites to a receptor domain. Binding of the ligand directs the folding of a mutually exclusive secondary structural switch in a downstream regulatory domain that directly interfaces with the expression machinery (either RNA polymerase or the ribosome). A variety of basic metabolic pathways including purine, amino acid, and cofactor biosynthesis in a number of bacteria, including Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis;in Bacillus and related Gram-positive species, over 4% of all genes are controlled in this fashion. Since these RNAs have already evolved to specifically bind small molecules, they have become of great interest as novel targets for designing therapeutics targeted against pathogenic bacteria. Towards the long-term goal of developing a molecular understanding of how riboswitches efficiently regulate gene expression, purine-binding riboswitches will be employed as a model system for addressing structural and mechanistic questions. This proposal details a set of specific aims that address: (1) how structural variation in the ligand receptor enables the regulatory response of individual riboswitches to be "tuned" to the gene it controls, (2) detail the structural plasticity of the RNA within the ligand binding site that allows it to be recognized by diverse purine analogs, (3) broaden our understanding into the mechanism by which ligand binding to the receptor is communicated to downstream regulatory domain to effect biological activity, and (4) determine the atomic-resolution structure of a riboswitch whose effector is coenzyme B12. To address these research goals, a combination of structural approaches including X-ray crystallography and small angle X-ray scattering, biochemical approaches such as chemical probing and transcriptional assays, and bioinformatics. The results of these proposed studies will serve to broaden our knowledge of RNA-based gene regulation as well as provide an atomic-level understanding of RNAs that are promising targets of antimicrobial agents.

Public Health Relevance

Riboswitches are a form of RNA-based gene regulation that is widely utilized in bacteria, including a number of medically important pathogenic bacteria such as S. aureus, M. tuberculosis, P. aeruginosa and Streptococcus species. Our work seeks to develop an atomic-level understanding of how these RNAs regulate bacterial through their ability to directly bind cellular metabolites. These studies serve to further our understanding into how RNA can be exploited as targets of antibacterial therapeutics via structure-based drug design.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM073850-09
Application #
8539005
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
2005-04-01
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
9
Fiscal Year
2013
Total Cost
$268,274
Indirect Cost
$85,673
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
80309
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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

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