Antisense-acting riboswitches: A poorly characterized yet important model of transcriptional regulation in prokaryotic organisms

Abstract

Riboswitches are RNA elements involved in regulating genes that participate in the biosynthesis or transport of essential metabolites. They are characterized by their ability to recognize their target molecules with high affinity and specificity. Riboswitches are commonly cotranscribed with their target genes and are located at the 5' end of their transcriptional units. To date, only two exceptional cases of riboswitches being situated at the 3' end and transcribing in the antisense direction of their regulated genes have been described. The first case involves a SAM riboswitch located at the 3' end of the ubiG-mccB-mccA operon in Clostridium acetobutylicum involved in converting methionine to cysteine. The second case concerns a Cobalamin riboswitch in Listeria monocytogenes that regulates the transcription factor PocR related to this organism's pathogenic process. In almost a decade since the first descriptions of antisense-acting riboswitches, no new examples have been described. In this work, we performed a computational analysis to identify new examples of antisense-acting riboswitches. We found 292 cases in which, according to the available information, we infer that the expected regulation of the riboswitch is consistent with the signaling molecule it senses and the metabolic function of the regulated gene. The metabolic implications of this novel type of regulation are thoroughly discussed.

Fig 1. Models of the putative regulatory mechanisms of RNA polymerases transcribing in opposite directions.

(A) RNA polymerase collision; (B) asRNA/mRNA duplex formation: (1) partially synthesized double-stranded RNA, (2) totally synthesized double-stranded RNA; (C) DNA supercoil accumulation. Representative elements are colored as follows: mRNA transcription complex, blue; asRNA transcription complex, pink; SD sequence, yellow. Created with BioRender.com.

Fig 2. Representative pathways of genes regulated by antisense-acting riboswitches.

(A) Gene encoding an enzyme involved in the interconversion of a compound related to the metabolite sensed by the riboswitch; (B) Gene encoding an enzyme that requires a cofactor sensed by the riboswitch to become active or participates in a metabolic pathway wherein another enzyme requires the cofactor; (C) Gene encoding a transporter protein for a compound related to the metabolite sensed by the riboswitch; (E) Gene that codes for a transcriptional regulator. Metabolic route active in the presence (yellow) or absence (green) of the metabolite sensed (red circle) by the antisense-acting riboswitch. Representative elements are colored as follows: Target genes, blue; antisense-acting riboswitch, violet. Enzymes are indicated by rectangles; compounds are indicated by ovals. Created with BioRender.com.

Fig 3. Representative syntenic context of genes regulated by antisense-acting riboswitches.

The upper panel on each section shows in detail an example of the different types of genes regulated by the antisense-acting riboswitches, while the lower panel shows the context in closely related organisms compared to the example. (A) Gene coding for a transposase: COG2138-Uncharacterized conserved protein, COG1648-Siroheme synthase, COG0733-Na+-dependent transporters of the SNF family, COG0739-Membrane proteins related to metalloendopeptidases; (B) Pseudogene: COG0041-Phosphoribosylcarboxyaminoimidazole (NCAIR) mutase, COG0026-Phosphoribosylaminoimidazole carboxylase (NCAIR synthetase), COG0015-Adenylosuccinate lyase; (C) Gene coding for an uncharacterized ORF with clear orthologs in multiple genomes; (D) Hypothetical small gene without orthologs: COG2261-Predicted membrane protein, COG0210-Superfamily I DNA and RNA helicases, CO1115-Na+/alanine symporter, COG0053-Predicted Co/Zn/Cd cation transporters. Representative elements are colored as follows: Riboswitch spuriously regulated genes, blue; antisense-acting riboswitch, violet; bona fide regulated gene, pink. Genes are colored according to the COG they belong to. Created with BioRender.com.