A Disconnect between High-Affinity Binding and Efficient Regulation by Antifolates and Purines in the Tetrahydrofolate Riboswitch

•Though most THF riboswitches contain two binding sites, one is not vital for function•The pABA moiety of THF greatly enhances regulatory activity, but not binding affinity•Some well-established antifolates can regulate the riboswitch’s activity•Adenine and derivatives exhibit “retro-binding” to the THF riboswitch

SummaryThe tetrahydrofolate (THF) riboswitch regulates folate transport and metabolism in a number of Firmicutes by cooperatively binding two molecules of THF. To further understand this riboswitch’s specificity for THF, binding and regulatory activity of a series of THF analogs and antifolates were examined. Our data reveal that although binding is dominated by the RNA’s interactions with the pterin moiety, the para-aminobenzoic acid (pABA) moiety plays a significant role in transcriptional regulation. Further, we find that adenine and several other analogs bind with high affinity by an alternative binding mechanism. Despite a similar affinity to THF, adenine is a poor regulator of transcriptional attenuation. These results demonstrate that binding alone does not determine a compound’s effectiveness in regulating the activity of the riboswitch—a complication in current efforts to develop antimicrobials that target these RNAs.