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ykkC riboswitches employ an add-on helix to adjust specificity for polyanionic ligands


The ykkC family of bacterial riboswitches combines several widespread classes that have similar secondary structures and consensus motifs but control different genes in response to different cellular metabolites. Here we report the crystal structures of two distinct ykkC riboswitches specifically bound to their cognate ligand ppGpp, a second messenger involved in stress response, or PRPP, a precursor in purine biosynthesis. Both RNAs adopt similar structures and contain a conserved core previously observed in the guanidine-specific ykkC riboswitch. However, ppGpp and PRPP riboswitches uniquely employ an additional helical element that joins the ends of the ligand-sensing domains and creates a tunnel for direct and Mg2+-mediated binding of ligands. Mutational and footprinting experiments highlight the importance of conserved nucleotides forming the tunnel and long-distance contacts for ligand binding and genetic response. Our work provides new insights into the specificity of riboswitches and gives a unique opportunity for future studies of RNA evolution.

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We thank D. Fenyo for help with bioinformatics searches. We thank personnel of beamlines 17-ID-2 at the Brookhaven National Laboratory and 24-ID at the Argonne National Laboratory for help with data collection. This research was supported by the NIH grants R01GM112940 to A.S., F31GM119357 and T32 GM88118 to A.P. This work uses the Northeastern Collaborative Access Team beamlines, which are funded by the National Institutes of Health (NIH) grants (P41 GM103403 and S10 RR029205) and resources of the Advanced Photon Source, operated for the DOE Office of Science under Contract no. DE-AC02-06CH11357. The beamline 17-ID-2 is supported in part by the DOE Office of Biological and Environmental Research (KP1605010, KC0401040) and by the NIH (P41GM111244).

Author information

A.P. crystallized the riboswitches, determined their structures and conducted biochemical experiments. A.S. contributed to determination and refinement of the structures. A.P. and A.S. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Correspondence to Alexander Serganov.

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Fig. 1: Overall structures and schematics of the ligand-bound S. lipocalidus PRPP and S. acidophilus ppGpp riboswitches.
Fig. 2: Structural elements of the PRPP and ppGpp riboswitches.
Fig. 3: Molecular details of the RNA–ligand recognition.
Fig. 4: Binding affinity of RNA–ligand interactions determined by ITC.
Fig. 5: Nuclease probing of the PRPP riboswitch RNA structure.
Fig. 6: Proposed mechanisms of the PRPP and ppGpp riboswitch action.