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The Impact of Second-Shell Nucleotides on Ligand Specificity in Cyclic Dinucleotide Riboswitches
Kathryn M Barth1,2, David A Hiller1,2, Scott A Strobel1,2
1Institute of Biomolecular Design and Discovery, Yale University, West Haven, Connecticut 06516, United States.
Biochemistry
|February 8, 2024
Summary
Riboswitches control gene expression by binding specific molecules. This study found that nucleotides outside the direct binding site can influence riboswitch specificity, even leading to promiscuous binding.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Riboswitches are genetic elements that regulate gene expression in response to small molecule ligands.
- Ligand specificity is crucial for riboswitch function, enabling discrimination between similar molecules.
- Variant riboswitches can exhibit distinct ligand specificities despite shared structural motifs.
Purpose of the Study:
- To investigate the molecular mechanisms underlying ligand specificity in cyclic dinucleotide riboswitches.
- To identify nucleotides beyond the immediate binding pocket that influence riboswitch ligand discrimination.
- To characterize a novel promiscuous riboswitch variant.
Main Methods:
- High-throughput mutational analysis using the SMARTT assay to assess thousands of potential regulatory sites.
- Structural analysis of riboswitch-ligand interactions.
- Quantitative assessment of ligand specificity and promiscuity.
Main Results:
- A mutated riboswitch variant exhibited promiscuity for both cyclic-di-GMP and cyclic-AMP-GMP, contrary to predictions.
- Nucleotides in both direct contact and distal regions (J1/2 linker, terminator helix) were found to impact ligand specificity.
- SMARTT analysis quantitatively mapped the effects of mutations on specificity and promiscuity.
Conclusions:
- Ligand specificity in riboswitches is not solely determined by nucleotides within the binding pocket.
- Distal nucleotides play a significant role in modulating riboswitch ligand recognition and specificity.
- These distal nucleotides offer potential targets for predicting or engineering riboswitch promiscuity.
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