Dual-acting riboswitch control of translation initiation and mRNA decay

Abstract

Riboswitches are mRNA regulatory elements that control gene expression by altering their structure in response to specific metabolite binding. In bacteria, riboswitches consist of an aptamer that performs ligand recognition and an expression platform that regulates either transcription termination or translation initiation. Here, we describe a dual-acting riboswitch from Escherichia coli that, in addition to modulating translation initiation, also is directly involved in the control of initial mRNA decay. Upon lysine binding, the lysC riboswitch adopts a conformation that not only inhibits translation initiation but also exposes RNase E cleavage sites located in the riboswitch expression platform. However, in the absence of lysine, the riboswitch folds into an alternative conformation that simultaneously allows translation initiation and sequesters RNase E cleavage sites. Both regulatory activities can be individually inhibited, indicating that translation initiation and mRNA decay can be modulated independently using the same conformational switch. Because RNase E cleavage sites are located in the riboswitch sequence, this riboswitch provides a unique means for the riboswitch to modulate RNase E cleavage activity directly as a function of lysine. This dual inhibition is in contrast to other riboswitches, such as the thiamin pyrophosphate-sensing thiM riboswitch, which triggers mRNA decay only as a consequence of translation inhibition. The riboswitch control of RNase E cleavage activity is an example of a mechanism by which metabolite sensing is used to regulate gene expression of single genes or even large polycistronic mRNAs as a function of environmental changes.

Fig. 1.

The E. coli lysC riboswitch modulates the mRNA level upon lysine binding. (A) Schematic representation of the predicted lysine riboswitch translational control. In the absence of lysine, the ON state exhibits an antisequestering stem, which exposes the RBS and allows translation initiation. However, when bound to lysine, the riboswitch adopts the OFF state in which the presence of a sequestering stem masks the RBS and inhibits gene expression. The region shared by the antisequestering stem (ON state) and the aptamer (OFF state) is represented in red. (B) β-Galactosidase assays of translational LysC-LacZ fusions for wild type and ON, OFF, and G31C mutants (Tables S1–S3). Enzymatic activities were measured in the absence and presence of 10 µg/mL lysine. Values were normalized to the enzyme activity obtained for the wild type in the absence of lysine. (C) β-Galactosidase assays of transcriptional lysC-lacZ fusions for the wild type and for the ON, OFF, and G31C mutants. Enzymatic activities were measured in the absence and presence of 10 µg/mL lysine. Values were normalized to the activity obtained for the wild type in the absence of lysine. (D) Northern blot analysis of lysC mRNA level. Wild-type E. coli strain MG1655 was grown to midlog phase in M63 minimal medium with 0.2% glucose at 37 °C, and total RNA was extracted at the indicated times immediately before (0−) and after (0+) the addition of lysine (10 µg/mL). The probe was designed to detect the riboswitch region (positions 42–201) of the lysC mRNA (SI Appendix, Fig. S4A). 16S rRNA was used as a loading control. (E) Northern blot analysis of the thiMD mRNA level. Bacterial growth culture and RNA extractions were performed as described in D. TPP was added at a final concentration of 500 µg/mL. The probe was designed to detect the riboswitch region (positions 4–141) of thiMD mRNA. 16S rRNA was used as a loading control. (F) Determination of the lysC mRNA stability. Bacterial growth culture and RNA extractions were performed as described in D, but without the addition of lysine. Rifampicin was added at a final concentration of 250 µg/mL. 16S rRNA was used as a loading control. (G) Determination of thiMD mRNA stability. Bacterial growth culture and RNA extractions were performed as described in D. Rifampicin was added at a final concentration of 250 µg/mL. 16S rRNA was used as a loading control.

Fig. 2.

RNase E and the RNA degradosome are involved in the rapid decrease of lysC mRNA. (A) Northern blot analysis of the lysC mRNA level in the context of the rne-131 strain. The E. coli strain rne-131 was grown to midlog phase in M63 minimal medium with 0.2% glucose at 37 °C, and total RNA was isolated at the indicated times immediately before (0−) and after (0+) the addition of lysine (10 µg/mL). 16S rRNA was used as a loading control. (B) Quantification analysis of Northern blots shown in A and Fig. 1D. The average values of three independent experiments with SDs are shown. (C) Northern blot analysis of the thiMD mRNA level in the context of the rne-131 strain. Bacterial growth culture and RNA extractions were performed as described in A. Total RNA was isolated at the indicated times immediately before (0−) and after (0+) the addition of TPP (500 µg/mL). 16S rRNA was used as a loading control. (D) Quantification analysis of Northern blots shown in C and Fig. 1E. The average values of three independent experiments with SDs are shown. (E) Northern blot analysis of lysC mRNA in the context of the rne-3071 [RNase E(TS)] strain grown at 30 °C (permissive temperature). The E. coli strain rne-3071 was grown to midlog phase in M63 minimal medium with 0.2% glucose at 30 °C, and total RNA was isolated at the indicated times immediately before (0−) and after (0+) the addition of lysine (10 µg/mL). 16S rRNA was used as a loading control. (F) Northern blot analysis of lysC mRNA in the context of the rne-3071 strain grown at 30 °C followed by a temperature shift at 44 °C (restrictive temperature). Bacterial growth culture and RNA extractions were performed as described in E. Total RNA was isolated at the indicated times immediately before (0−) and after (0+) the addition of lysine (10 µg/mL). Cells were incubated at 30 °C from 0–4 min and at 44 °C from 4–24 min. 16S rRNA was used as a loading control. Note that unprocessed 5S rRNA intermediates are detected at 44 °C, as is consistent with the inactivation of RNase E (26). (G) Quantification analysis of Northern blots shown in F and SI Appendix, Fig. S7B. The quantification represents the extent of recovery of lysC mRNA in the context of the rne-3071 strain when grown at 44 °C. Such a recovery is not observed in wild-type cells. The average values of three independent experiments with SDs are shown.

Fig. 3.

The RNase E cleavage sites are located in the lysC riboswitch expression platform. (A) Predicted secondary structure of the lysC riboswitch in the presence of lysine (OFF state). Nucleotides shared by the antisequestering stem (ON state) and the aptamer (OFF state) are shown in red. RNase E cleavage Site1 (positions 249–250) and Site2 (positions 257–258) determined in vitro are indicated by arrows. Identical sequences surrounding both cleavage sites are in orange boxes. The RBS and AUG start codon are in blue boxes. (B) Predicted secondary structure of the antisequestering stem of the lysC riboswitch (positions 182–277). RNase E cleavage sites 1 and 2 are shown by arrows. (C) In vitro mapping of the RNA degradosome cleavage sites using 3′-end–radiolabeled riboswitch expression platform (positions 212–309). Increasing amounts of purified RNA degradosomes (0, 0.5, 0.76, 1.0, and 1.5 ng/µL) were used in the cleavage buffer (lanes 4–8). RNase TA (lane 1), RNase T1 (lane 2), and alkaline hydrolysis (L; lane 3) were used to generate molecular markers for gel migration. (D) In vitro mapping of the RNA degradosome cleavage sites using wild-type (lanes 5–8) or G31C riboswitch mutant (lanes 9–12). Riboswitch molecules were incubated in the absence and in the presence of lysine (68 µM) and/or RNA degradosomes (1 ng/µL). Reaction products were analyzed by reverse transcription using the EM1444 oligonucleotide (SI Appendix, Table S2). Lanes 1–4 represent the sequencing ladder. (E) In vitro mapping of the RNA degradosome cleavage sites using the OFF state riboswitch mutant in the absence (lane 5) and presence (lane 6) of RNA degradosomes (1 ng/µL). Cleavage sites were detected using reverse transcription as indicated in D. See SI Appendix, Fig. S2 for details about the OFF state riboswitch mutant. (F) In vitro mapping of the RNA degradosome cleavage sites using the ON state riboswitch mutant in the absence (lane 5) and presence (lane 6) of RNA degradosomes (1 ng/µL). Cleavage sites were detected using reverse transcription as indicated in D. See SI Appendix, Fig. S2 for details about the ON state riboswitch mutant. (G) In vitro mapping of the RNA degradosome cleavage sites using the ∆Site1-2 (lanes 1–4) and the ∆Site1-2/OFF state (lanes 9 and 10) riboswitch mutants. Experiments were performed in the absence and presence of lysine (68 µM) and/or RNA degradosomes (1 ng/µL). Cleavage sites were detected using reverse transcription as indicated in D. The expected positions of cleaved products are indicated on the right of the gel by asterisks. See SI Appendix, Fig. S2 for details about the ∆Site1-2 riboswitch mutant. The ∆Site1-2/OFF construct contains mutations used to obtain the ∆Site1-2 and OFF riboswitch mutants. Note that the gels shown for ∆Site1-2 and ∆Site1-2/OFF cleavage reactions were taken from different experiments.

Fig. 4.

The lysC riboswitch can be directed to control either translation initiation or mRNA decay. (A) β-Galactosidase assays using translational (trL) and transcriptional (trX) fusions of the lysC riboswitch for the wild-type, ∆Site1, ∆Site2, and ∆Site1-2 constructs. β-Galactosidase enzymatic activities were measured in the absence and presence of 10 µg/mL lysine. Values obtained for translational and transcriptional fusions were normalized to enzymatic activities obtained for WT translational and transcriptional fusions, respectively, in the absence of lysine. See SI Appendix, Fig. S2 for details about riboswitch mutant constructs. (B) β-Galactosidase assays using translational and transcriptional fusions of the lysC riboswitch for the wild-type, AAG, and GACG constructs. Values obtained for translational and transcriptional fusions were normalized to enzymatic activities obtained for WT translational and transcriptional fusions, respectively, in the absence of lysine. See SI Appendix, Fig. S2 for details about riboswitch mutant constructs.

Fig. 5.

Deletion of Site1 affects the lysine-dependent turnover of the lysC mRNA. (A) Northern blot analysis of the lysC mRNA expressed from a pBAD-lysC plasmid in the context of a ΔlysC strain. The plasmid was transformed into a strain in which the endogenous lysC gene was deleted (EM1055∆lysC::cat; SI Appendix, SI Materials and Methods). The resulting MPC70 strain was grown at 37 °C to midlog phase in M63 medium with 0.2% glycerol. Total RNA extractions were performed 15 min (lane 15−) before lysC mRNA induction with 0.1% arabinose (Ara) and before (lane 0−) and after (0+) the addition of lysine (10 μg/mL). The probe was designed to detect the riboswitch region (positions 42–201) of the lysC mRNA. 16S rRNA was used as a loading control. (B) Northern blot analysis of the lysC ∆Site1 mRNA expressed from a pBAD-lysC-∆Site1 plasmid. Bacterial growth cultures and RNA extractions were performed as described in A. 16S rRNA was used as a loading control. (C) Quantification analysis of Northern blots shown in A and B. The average values of three independent experiments are shown with SD.

Fig. 6.

Regulation mechanisms of riboswitches controlling at the levels of translation initiation and mRNA decay. (A) Regulation mechanism of btuB and thiM riboswitches controlling translation initiation (nonnucleolytic repression). In the absence of ligand, the riboswitch adopts the ON state in which translation initiation is allowed and gene expression ensues. However, ligand binding to the riboswitch leads to the adoption of the OFF state in which the RBS is sequestered, resulting in the inhibition of translation initiation and ultimately to mRNA degradation. (B) Regulation mechanisms of the lysC riboswitch controlling translation initiation and mRNA decay (nucleolytic repression). In the absence of ligand, the riboswitch folds into the ON state that sequesters the RNase E cleavage site (indicated by a star) and that allows translation initiation for gene expression. Upon ligand binding, the riboswitch adopts the OFF state that exposes an RNase E cleavage site and sequesters the RBS, both ensuring definitive gene repression.

Fig. P1.

Regulatory mechanisms of the lysC riboswitch controlling translation initiation and mRNA decay. In the absence of lysine (Lys), the riboswitch folds into the ON state, exposing the RBS to allow translation initiation and lysC expression. In this ON state conformation, the riboswitch also sequesters an RNase E cleavage site (indicated by a star) to prevent mRNA decay from the RNase E-containing RNA degradosome. However, upon lysine binding, the riboswitch folds into an OFF state that both sequesters the RBS and reveals the RNase E cleavage site, ensuring efficient genetic repression and mRNA decay. The lysC initiation codon and ORF are denoted by “AUG” and “lysC ORF,” respectively.