Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 292, Issue 28, Pages 11650-11658Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M117.787176
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Funding
- National Institutes of Health [R01 GM073850]
- National Institutes of Health Biophysics Training Grant [T32 GM065103]
- National Institutes of Health Ruth L. Kirschstein Fellowship [F32 GM095121]
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Riboswitches are a widely distributed class of regulatory RNAs in bacteria that modulate gene expression via small-molecule-induced conformational changes. Generally, these RNA elements are grouped into classes based upon conserved primary and secondary structure and their cognate effector molecule. Although this approach has been very successful in identifying new riboswitch families and defining their distributions, small sequence differences between structurally related RNAs can alter their ligand selectivity and regulatory behavior. Herein, we use a structure-based mutagenic approach to demonstrate that cobalamin riboswitches have a broad spectrum of preference for the two biological forms of cobalamin in vitro using isothermal titration calorimetry. This selectivity is primarily mediated by the interaction between a peripheral element of the RNA that forms a T-loop module and a subset of nucleotides in the cobalamin-binding pocket. Cell-based fluorescence reporter assays in Escherichia coli revealed that mutations that switch effector preference in vitro lead to differential regulatory responses in a biological context. These data demonstrate that a more comprehensive analysis of representative sequences of both previously and newly discovered classes of riboswitches might reveal subgroups of RNAs that respond to different effectors. Furthermore, this study demonstrates a second distinct means by which tertiary structural interactions in cobalamin riboswitches dictate ligand selectivity.
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