A eubacterial riboswitch class that senses the coenzyme tetrahydrofolate

Abstract

Comparative sequence analyses of bacterial genomes are revealing many structured RNA motifs that function as metabolite-binding riboswitches. We have identified an RNA motif frequently positioned in the 5' UTRs of folate transport and biosynthesis genes in Firmicute genomes. Biochemical experiments confirm that representatives of this new-found RNA class selectively bind derivatives of the vitamin folate, including di- and tetrahydrofolate coenzymes. In addition, representatives of this aptamer class occasionally reside upstream of RNA structures that are predicted to control translation initiation in response to ligand binding. These findings expand the number of coenzymes that are directly sensed by RNA and reveal possible riboswitch-controlled regulons that respond to changes in single-carbon metabolism.

Figure 1. Conserved RNA motif associated with genes for folate metabolism

(A) Consensus sequence and secondary structure model for the candidate riboswitch aptamer. See Supplemental Information for the alignment and methods used to establish nucleotide conservation and covariation. (B) Chemical structures of folate, DHF, THF and other folate derivatives used in this study. (C) Folate biosynthesis and uptake pathway. Genes under control of a THF riboswitch are highlighted with relative frequency of regulatory interaction reflected by the heights of the blue bars. GTP is guanosine-5′-triphosphate; PABA is p-aminobenzoic acid; Glu is L-glutamine.

Figure 2. Evidence for THF riboswitch function

(A) Sequence and predicted secondary structure of the 106 folT RNA from A. metalliredigens. Nucleotides shaded yellow, red or green identify internucleotide linkages that undergo constant, decreasing or increasing scission when THF is added to an in-line probing assay (see gel image in B). Asterisks identify the 5′ and 3′ boundaries of the in-line probing annotations, which are restricted due to limitations of product separation by polyacrylamide gel electrophoresis. Shaded boxes designate sequence changes present in mutants M1 through M4 as indicated. M5 construct is truncated at the bracket. Lowercase letters identify nucleotides added to the construct to facilitate efficient in vitro transcription. (B) In-line probing analysis of the A. metalliredigens 106 folT RNA. NR, T1 and ⁻OH designate precursor (Pre) RNAs subjected to no reaction, partial digestion with RNase T1 (cleaves after G residues), and partial digest under alkaline conditions (cleaves at every position). Other lanes include Pre RNAs subjected to in-line probing reactions without THF (–) or with increasing concentrations of THF (1 nM to 10 μM). Vertical lines identify cleavage products whose yields are altered by THF addition, and numbered regions were quantified and used to estimate K_D values. (C) Plot of the fraction of RNAs bound to ligand versus the logarithm of the concentration of THF. The solid line represents a theoretical binding curve for a one-to-one interaction with a K_D of 70 nM. Fraction bound values were generated by quantifying band intensities from the regions identified in B. (D) A possible THF riboswitch expression platform is evident in the L. casei folT mRNA. The nucleotides shaded in orange can form an alternative base pairing interaction in the absence of THF that may interfere with the formation of the putative anti-RBS hairpin.