The yjdF riboswitch candidate regulates gene expression by binding diverse azaaromatic compounds

Abstract

The yjdF motif RNA is an orphan riboswitch candidate that almost exclusively associates with the yjdF protein-coding gene in many bacteria. The function of the YjdF protein is unknown, which has made speculation regarding the natural ligand for this putative riboswitch unusually challenging. By using a structure-probing assay for ligand binding, we found that a surprisingly broad diversity of nitrogen-containing aromatic heterocycles, or "azaaromatics," trigger near-identical changes in the structures adopted by representative yjdF motif RNAs. Regions of the RNA that undergo ligand-induced structural modulation reside primarily in portions of the putative aptamer region that are highly conserved in nucleotide sequence, as is typical for riboswitches. Some azaaromatic molecules are bound by the RNA with nanomolar dissociation constants, and a subset of these ligands activate riboswitch-mediated gene expression in cells. Furthermore, genetic elements most commonly adjacent to the yjdF motif RNA or to the yjdF protein-coding region are homologous to protein regulators implicated in mitigating the toxic effects of diverse phenolic acids or polycyclic compounds. Although the precise type of natural ligand sensed by yjdF motif RNAs remains unknown, our findings suggest that this riboswitch class might serve as part of a genetic response system to toxic or signaling compounds with chemical structures similar to azaaromatics.

Riboswitch diversity and distribution

“…Members of the second riboswitch class respond to a diverse collection of multi-ring molecules that can best be called "azaaromatic" ligands, and thus these RNAs are called azaaromatic riboswitches (Li et al 2016). Within the genomic DNA database used for our study, 743 representatives of azaaromatic riboswitches were identified, which suggests that members of this class sense a natural compound that is of wide importance to bacteria.…”

Former orphan riboswitches reveal unexplored areas of bacterial metabolism, signaling, and gene control processes

“…For the first approach, extremely rare gene associations were considered, as they hinted that the ligand might have some connection to nicotinamide adenine dinucleotide (NAD + ) or FMN metabolism. These coenzymes and their derivatives were examined for in vitro binding to yjdF RNAs, which unexpectedly revealed that diverse flavin analogs are bound by yjdF RNAs with relatively high affinity (Li et al 2016). Mutations to highly conserved nucleotides disrupt Cold Spring Harbor Laboratory Press on July 31, 2020 -Published by rnajournal.cshlp.org Downloaded from associations with these ligands, indicating that this promiscuous binding was unlikely to be due to non-specific interactions.…”

“…Specifically, a B. subtilis riboswitch-reporter strain was grown in ~2,000 conditions sampled by a growth media library. Only five supplemented compounds triggered the yjdF RNA-lacZ reporter, all of which were large, planar, nitrogenous, polycyclic molecules (Li et al 2016). The hits from the screen, as well as the flavin derivatives identified through additional binding assays, share little resemblance with one another except that they all fall into the general category of 'azaaromatic' molecules.…”

Applications and limitations of regulatoryRNAelements in synthetic biology and biotechnology

“…) and azaaromatic compounds (Li et al . ), a long lag could be harmful to the cell. Furthermore, it has been shown that aptamer folding depends on physiological conditions (Chen et al .…”