Riboswitches in eubacteria sense the second messenger c-di-AMP

Abstract

Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered bacterial second messenger implicated in the control of cell wall metabolism, osmotic stress responses and sporulation. However, the mechanisms by which c-di-AMP triggers these physiological responses have remained largely unknown. Notably, a candidate riboswitch class called ydaO associates with numerous genes involved in these same processes. Although a representative ydaO motif RNA recently was reported to weakly bind ATP, we report that numerous members of this noncoding RNA class selectively respond to c-di-AMP with subnanomolar affinity. Our findings resolve the mystery regarding the primary ligand for this extremely common riboswitch class and expose a major portion of the super-regulon of genes that are controlled by the widespread bacterial second messenger c-di-AMP.

Figure 1. Binding of c-di-AMP by a ydaO motif RNA

a, Chemical structure of c-di-AMP. b, Consensus sequence and secondary structure of ydaO motif RNAs derived from ~ 3,000 examples. Red, black and gray nucleotides, respectively, are present in greater than 97, 90 and 75% of the representatives. Predicted base-paired substructures are labeled P1 through P7, with one pseudoknot as indicated. Green shading indicates phylogenetic evidence of base pairing. Other annotations are as described previously. c, Structural modulation of the WT 165 ydaO RNA from B. subtilis. Highlighted nucleotides indicate locations of changes in spontaneous cleavage upon addition of c-di-AMP, mapped using the data in d and Supplementary Fig. 3. d, Polyacrylamide gel electrophoresis (PAGE) analysis of an in-line probing assay with 165 ydaO RNA exposed to various concentrations of c-di-AMP (100 pM to 10 μM in half-log intervals) or ATP (17.8 μM to 3.16 mM in quarter-log intervals). NR, T1 and ⁻OH, respectively, designate no reaction, partial digestion with either RNase T1 (cleaves after guanosine nucleotides) or hydroxide ions (cleaves after any nucleotide). Precursor RNA (Pre) and certain RNase T1 cleavage product bands are identified. Locations of spontaneous RNA cleavage changes brought about by c-di-AMP (regions 1 through 6) are identified by asterisks (see Supplementary Fig. 14 for the full length gel). e, Plot of the fraction of riboswitch RNA bound to ligand versus the logarithm of the molar concentration of c-di-AMP as inferred from the modulation of spontaneous cleavage products in d.

Figure 2. Ligand binding characteristics of WT and variant 165 ydaO RNAs

a, Plot of the K_D values for the binding of c-di-AMP analogs by WT 165 ydaO RNA. A 5′PS is adenosine 5′-phosphosulfate, IMP is inosine 5′-phosphate, c-pGpA is the cyclic dinucleotide composed of one GMP and one AMP (also called cGA), and c-di-dAMP is the DNA analog of c-di-AMP. Gray circle and arrow indicates that the compounds listed exhibit no detectable binding at this concentration. Open circle and arrow indicates the K_D for c-di-AMP is no poorer than this value. b, Various mutant constructs examined for ligand binding. Nucleotide changes made at the sites annotated M1 through M5 are boxed. Red letters identify highly conserved nucleotides. c, In-line probing analyses of WT and M1 through M4 RNAs as defined in b in the absence (−) or presence of 10 nM c-di-AMP. Gel images depict spontaneous RNA cleavage patterns encompassing sites 1 and 2 as defined in Fig. 1c (see Supplementary Fig. 15 for the full length gel). d, In-line probing analysis of M5 in the absence (−) of ligand, or the presence of c-di-AMP (10, 32 or 100 nM), or ATP (1 mM). Gel images depict the region encompassing sites 1 through 6 with annotations as described for Fig. 1d. The arrow identifies nucleotides in the loop region of P7 that become unstructured on ligand binding, as expected since their complementary sequences have been removed in this construct (see Supplementary Fig. 16 for the full length gel).

Figure 3. Riboswitch regulation of gene expression by c-di-AMP

a, PAGE analysis of an in vitro transcription termination assay using the yuaA riboswitch from B. subtilis. T is the riboswitch-terminated RNA transcript and FL is the full-length run-off transcript. M is a marker lane comprising the transcription products from a similar DNA template encoding the riboswitch plus six additional nucleotides beyond the predicted terminator site (see Supplementary Fig. 17 for the full length gel). b, Reporter gene construct used to assess regulation by c-di-AMP riboswitches. P is the native ydaO promoter, lacZ is the reporter gene, and RBS is the ribosome binding site. The predicted intrinsic transcription terminator stem for the B. subtilis ydaO riboswitch is shown in detail. c, Plot of reporter gene expression for various riboswitch constructs and genetic backgrounds (normal is the YP79 strain of B. subtilis) normalized to the level of expression observed with the WT riboswitch construct. Error bars represent the standard deviation of three replicate experiments conducted on three different days.

Figure 4. The super-regulon for second messenger signaling through c-di-AMP riboswitches

Bacterial lineages presented are Actinobacteria (Act), Bacillales (Bac), Clostridia, (Clo) and Cyanobacteria (Cya).