Riboswitches. A riboswitch-containing sRNA controls gene expression by sequestration of a response regulator

Abstract

The ethanolamine utilization (eut) locus of Enterococcus faecalis, containing at least 19 genes distributed over four polycistronic messenger RNAs, appears to be regulated by a single adenosyl cobalamine (AdoCbl)-responsive riboswitch. We report that the AdoCbl-binding riboswitch is part of a small, trans-acting RNA, EutX, which additionally contains a dual-hairpin substrate for the RNA binding-response regulator, EutV. In the absence of AdoCbl, EutX uses this structure to sequester EutV. EutV is known to regulate the eut messenger RNAs by binding dual-hairpin structures that overlap terminators and thus prevent transcription termination. In the presence of AdoCbl, EutV cannot bind to EutX and, instead, causes transcriptional read through of multiple eut genes. This work introduces riboswitch-mediated control of protein sequestration as a posttranscriptional mechanism to coordinately regulate gene expression.

Fig. 1. EA and AdoCbl induce eut gene expression

By RNA-seq, all the eut genes were expressed in the presence of EA and AdoCbl, whereas little or no expression was observed in minimal medium.

Fig. 2. AdoCbl binding to the riboswitch causes premature termination within a sRNA, preventing generation of the P3/P4 hairpins that bind EutV

(A) Location of ³²P-radiolabeled RNA anti-sense probes used to analyze the eutT-eutG intergenic region. (B) Total RNA was isolated from cells grown in the indicated medium conditions, resolved by denaturing polyacrylamide gel electrophoresis, transferred to a membrane, and hybridized with different antisense probes (1, 2 and 3). (C) The mapped 5′ and 3′ termini and the M3 mutation are indicated on a secondary structure depiction of EutX. A putative intrinsic terminator is highlighted by a gray-shaded box. (D) In vitro transcription termination assays with wild-type EutX template display a premature termination product only in the presence of AdoCbl. Termination is lost in the M3 mutant template. (E) EMSA of binding of EutV to EutX. A 117-nt fragment of EutX (117EutX) and EutVD54E protein [constitutively active for RNA binding (5)] were used. Controls included addition of 30 μM of an unlabeled competitor P1/P2 RNA and an unrelated RNA. (F) DRaCALA to assess EutV binding to the 117EutX RNA by using the same reaction conditions and control RNAs as in (E). (G) EMSA to investigate whether EutX could compete for EutV binding to P1/P2.

Fig. 3. Both the riboswitch and the P3/P4 hairpins are required for inducible expression in vivo

(A) A diagram of the experimental design. A plasmid containing a eutG-lacZ fusion was manipulated to contain a deletion of the riboswitch, the P3/P4 hairpin, or both. The plasmid was introduced into E. faecalis strains containing either wild-type EutX or a deletion. (B) β-Galactosidase assays of strains shown in (A) when grown in modified minimal medium containing EA or both EA and AdoCbl. (C) β-Galactosidase assays of strains missing EutX but carrying eutG-lacZ plasmids that contain either the M1 or M2 mutations. The M1 mutations are A249U and G252U and the M2 mutations are A273U and G276U site-directed changes. The data are presented as the average of three or more independent experiments, and the error bars represent the standard deviation.