Biochemical validation of a second class of tetrahydrofolate riboswitches in bacteria

We previously reported a large collection of structured noncoding RNAs (ncRNAs) that includes many riboswitch candidates identified through comparative sequence analysis of bacterial intergenic regions. One of these candidates, initially named the “folE motif,” adopts a simple architecture commonly found upstream of folE genes. FolE enzymes catalyze the first enzyme in the de novo folate biosynthesis pathway. Herein, we demonstrate that folE motif RNAs selectively bind the enzyme cofactor tetrahydrofolate (THF) and several of its close derivatives. These aptamers, commonly found in Gram-negative bacteria, are distinct from aptamers of the previous validated THF riboswitch class found in Gram-positive bacteria. Our findings indicate that folE motif RNAs are aptamer domains for a second THF riboswitch class, named THF-II. The biochemical validation of THF-II riboswitches further highlights the ability of bacteria to utilize diverse RNA structures to sense universal enzyme cofactors that are predicted to be of ancient origin.