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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063162-10
Application #
8451334
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter C
Project Start
2001-04-01
Project End
2016-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
10
Fiscal Year
2013
Total Cost
$341,709
Indirect Cost
$98,062
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
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Dutta, Debapratim; Wedekind, Joseph E (2015) Gene Regulation Gets in Tune: How Riboswitch Tertiary-Structure Networks Adapt to Meet the Needs of Their Transcription Units. J Mol Biol 427:3469-72
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Liberman, Joseph A; Suddala, Krishna C; Aytenfisu, Asaminew et al. (2015) Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics. Proc Natl Acad Sci U S A 112:E3485-94
Aytenfisu, Asaminew H; Liberman, Joseph A; Wedekind, Joseph E et al. (2015) Molecular mechanism for preQ1-II riboswitch function revealed by molecular dynamics. RNA 21:1898-907
Suddala, Krishna C; Walter, Nils G (2014) Riboswitch structure and dynamics by smFRET microscopy. Methods Enzymol 549:343-73
Liberman, Joseph A; Bogue, Jarrod T; Jenkins, Jermaine L et al. (2014) ITC analysis of ligand binding to preQ₁ riboswitches. Methods Enzymol 549:435-50
Heldenbrand, Hugh; Janowski, Pawel A; GiambaÅŸu, 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
Suddala, Krishna C; Rinaldi, Arlie J; Feng, Jun et al. (2013) Single transcriptional and translational preQ1 riboswitches adopt similar pre-folded ensembles that follow distinct folding pathways into the same ligand-bound structure. Nucleic Acids Res 41:10462-75

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