View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Chemistry & Biology Brief Communication A Eubacterial Riboswitch Class That Senses theCoenzymeTetrahydrofolate Tyler D. Ames,1 Dmitry A. Rodionov,2,3 Zasha Weinberg,4 and Ronald R. Breaker1,4,5,* 1Department of Molecular, Cellular and Developmental Biology, Yale University, Box 208103, New Haven, CT 06520-8103, USA 2Institute for Information Transmission Problems, Russian Academy of Sciences, Moscow 127994, Russia 3Burnham Institute for Medical Research, La Jolla, CA 92037, USA 4Howard Hughes Medical Institute, Chevy Chase, MD 20815-6789, USA 5Department of Molecular Biophysics and Biochemistry, Yale University, Box 208103, New Haven, CT 06520-8103, USA *Correspondence: [email protected] DOI 10.1016/j.chembiol.2010.05.020 SUMMARY in metagenomic DNA sequences (Rodionov et al., 2003; Barrick et al., 2004; Weinberg et al., 2007; Yao et al., 2007; Rodionov, Comparative sequence analyses of bacterial 2007). In the current study, we describe the discovery and genomes are revealing many structured RNA motifs characterization of a novel riboswitch candidate that is that function as metabolite-binding riboswitches. frequently associated with genes whose protein products We have identified an RNA motif frequently posi- transport folate or catalyze reactions related to single-carbon tioned in the 50 UTRs of folate transport and biosyn- metabolism. thesis genes in Firmicute genomes. Biochemical RESULTS AND DISCUSSION experiments confirm that representatives of this new-found RNA class selectively bind derivatives of Our bioinformatics pipeline identified 57 distinct representatives the vitamin folate, including di- and tetrahydrofolate (see Figure S1 available online) of a novel RNA motif (Figure 1A) coenzymes. In addition, representatives of this ap- consisting of four base-paired regions (P1 through P4) and an tamer class occasionally reside upstream of RNA additional base-paired pseudoknot structure. The motif is structures that are predicted to control translation frequently located immediately upstream of bacterial folate (Fig- initiation in response to ligand binding. These find- ure 1B) uptake transporter folT genes (Figure 1C; Figure S2), and ings expand the number of coenzymes that are in some cases upstream of the folate biosynthesis genes folE, directly sensed by RNA and reveal possible ribo- folC, and folQPBK (Table S1). The RNA motif was found both switch-controlled regulons that respond to changes in complete genomes (Clostridiales and Lactobacillales orders) in single-carbon metabolism. and in metagenome (human gut) data. We hypothesized that the RNA may be a riboswitch that binds to a compound related to folate. INTRODUCTION Folates are composed of three moieties: pterin, p-aminoben- zoate, and glutamate (Figure 1B). An active form of folate, tetra- Metabolite-binding riboswitches are gene control elements con- hydrofolate (THF), is an essential cofactor in one-carbon transfer structed entirely of RNA (Roth and Breaker, 2009). Each usually reactions of central metabolism. De novo THF biosynthesis is composed of an aptamer domain that binds a specific metab- starts from GTP and proceeds in seven consecutive steps (de olite and an expression platform that transforms an aptamer Cre´ cy-Lagard et al., 2007)(Figure 1C). Alternatively, Gram-posi- binding event into an adjustment in the level of expression of tive bacteria salvage folate from the environment utilizing target genes. Riboswitches are most commonly found in the 50 a unique energy-coupling factor (ECF) transport system com- untranslated regions (UTRs) of bacterial mRNAs, where they posed of a folate-binding protein (FolT) and a common ECF usually control gene expression by influencing transcription module (Rodionov et al., 2009). The identified riboswitch candi- termination or translation initiation (Barrick and Breaker, 2007; date is predicted to control expression of folT in many Firmi- Dambach and Winkler, 2009). Riboswitches also have been cutes, including Lactobacillus species which are auxotrophic shown to act in trans by base pairing with mRNAs transcribed for folate. There is a single example, occurring in Ruminococcus elsewhere in the genome (Loh et al., 2009). Additionally, there obeum, in which the RNA motif is associated with the folate are experimentally validated examples of eukaryotic ribo- biosynthesis folEQPBK operon. switches in both fungi (Cheah et al., 2007) and plants (Bocobza A 106 nucleotide RNA construct termed 106 folT (Figure 2A) et al., 2007; Croft et al., 2007; Wachter et al., 2007) that modulate that encompasses a representative motif located in the 50 UTR protein levels by controlling mRNA splicing. of the folT gene from Alkaliphilus metalliredigens was prepared Currently, the most productive methods for finding novel for analysis. This RNA was subjected to in-line probing (Soukup riboswitch classes make use of computer-aided searches to and Breaker, 1999; Regulski and Breaker, 2008) to assess ligand identify intergenic regions that are highly conserved in primary binding and structural modulation characteristics. In-line prob- and secondary structure among several bacterial genomes or ing assays take advantage of the inherent instability of RNA Chemistry & Biology 17, 681–685, July 30, 2010 ª2010 Elsevier Ltd All rights reserved 681 Chemistry & Biology Tetrahydrofolate Riboswitches O Figure 1. Conserved RNA Motif Associated O A pseudoknot B H with Genes for Folate Metabolism G R O G N (A) Consensus sequence and secondary structure G G U folate H Y U G H model for the candidate riboswitch aptamer. See O N A 10 Supplemental Information for the alignment and R Y 5 N O O methods used to establish nucleotide conserva- G P3 N Y pteroyl glutamyl tion and covariation. R N N (B) Chemical structures of folate, DHF, THF, and R H2 N O R J3/4 8 O other folate derivatives used in this study. R = A or G Y A P4 H Y G O (C) Folate biosynthesis and uptake pathway. Y = C or U G U C AA R N G R R dihydrofolate H Genes under control of a THF riboswitch are high- A (DHF) H lighted with relative frequency of regulatory inter- Y Y O N variable A Y action reflected by the heights of the blue bars. G N O O 0 U C N GTP is guanosine-5 -triphosphate; PABA is p-ami- G G J4/2 covarying J2/3 C R nobenzoic acid; Glu is L-glutamine. G U H N N N O mutations 2 H O Y R H R Y G G R G U O tetrahydrofolate N compatible P2 H C Y U A C G (THF) H mutations O N take on a puckered configuration (Poe H nucleotide N O O and Hoogsteen, 1978) that may also identity G Y N serve as a molecular recognition feature. R Y H N 97% G N N Since these aptamers tolerate modifica- A C H2 N N 90% A H J1/2 U tions at the N5 and N10 positions of the N 75% G U O Y ligand, they are unlikely to be important A C J2/1 nucleotide G C N H N molecular recognition contacts to these present R U H positions. Given the preferential affinity 97% 75% P1 C G N N for the coenzyme forms of folate, we 90% 50% 5‘ 5,10-methylene THF 10-formyl THF speculate that THF, or perhaps one or more of its single-carbon derivatives, is C folE folQ folB folK folP folC folA the biologically relevant ligand for these GTP DHF THF aptamers. PABA Glu Regions of the RNA that are particularly folA susceptible to spontaneous cleavage during in-line probing reactions (Fig- folT transport Folate ure 2B) largely correspond to nucleotides predicted to reside in single-stranded regions (Figure 2C). Furthermore, sites that undergo ligand-dependent reduction phosphoester bonds located in unstructured regions to reveal in spontaneous cleavage carry highly conserved nucleotides. folding changes caused by ligand binding. The 106 folT RNA These results are consistent with our secondary structure model undergoes extensive changes in RNA structure (Figure 2B) as for the aptamer and suggest that ligand binding stabilizes it binds THF with an apparent dissociation constant (KD)of a tertiary structure formed in part by the nucleotides whose 70 nM (Figure 2C). Two other folate derivatives, dihydrofolate base identities are well conserved. (DHF) and tetrahydrobiopterin, also bind with KD values of To further test the validity of our model, RNA constructs con- 300 nM (Figure S3). However, no structural modulation was taining three consecutive mutations that disrupt the P2 (con- observed when the RNA was incubated with folate, pterin, struct M1) or P3 (M2) stems of the 106 folT RNA (Figure 2A) p-aminobenzoate, glutamate, or p-aminobenzoyl glutamate at were examined for THF binding using in-line probing assays. concentrations as high as 1 mM. Both M1 and M2 constructs are less structured than the wild- Similar in-line probing results with THF and DHF were type (WT) construct, as indicated for M1 by an increase in spon- observed using several other representatives from this RNA taneous cleavage at phosphodiester linkages of the nucleotides class (data not shown). Additional assays revealed that these that comprise the stems (Figure S4). Furthermore, the KD of M1 RNAs also bind to various N5- and N10-modified THF deriva- for THF is 1000-fold poorer compared with WT, whereas the tives, including 5-formyl THF, 5-methyl THF, 10-formyl THF, M2 mutant does not bind to THF at concentrations as high as 5,10-methenyl THF, and 5,10-methylene THF. These results indi- 3.16 mM. In contrast, RNA constructs carrying an additional cate that the new-found RNAs function as selective aptamers for three mutations that restore base pairing to the P2 (M3) or P3 specific reduced forms of the vitamin folate. The ability of the (M4) stems (Figure 2A) exhibit KD values comparable to that aptamers to discriminate between folate and the reduced deriv- observed for WT.