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Bacterial small RNAs (sRNAs) are well known to modulate gene expression by base pairing with trans-encoded transcripts and are typically non-coding. However, several sRNAs have been reported to also contain an open reading frame and thus are considered dual-function RNAs. In this study, we discovered a dual-function RNA from Vibrio cholerae, called VcdRP, harboring a 29 amino acid small protein (VcdP), as well as a base-pairing sequence. Using a forward genetic screen, we identified VcdRP as a repressor of cholera toxin production and link this phenotype to the inhibition of carbon transport by the base-pairing segment of the regulator. By contrast, we demonstrate that the VcdP small protein acts downstream of carbon transport by binding to citrate synthase (GltA), the first enzyme of the citric acid cycle. Interaction of VcdP with GltA results in increased enzyme activity and together VcdR and VcdP reroute carbon metabolism. We further show that transcription of vcdRP is repressed by CRP allowing us to provide a model in which VcdRP employs two different molecular mechanisms to synchronize central metabolism in V. cholerae.

Non-coding RNAs (ncRNAs) and small proteins have both emerged as important regulators of gene expression. Dual-function RNAs encode a small protein and have a separate function as a regulatory RNA. Although first discovered in bacteria, dual-function RNAs have now been identified and characterized in eukaryotes as well. These RNAs allow two activities of a single gene to regulate targets at multiple levels. The work described here explores how two novel and one synthetic dual-function RNA act and how competition between the components of a dual-function RNA impacts their functions. AzuCR is a 164-nucleotide E. coli RNA that was previously shown to encode a 28 amino acid protein (AzuC). This work demonstrates that the AzuC small protein impacts glycerol metabolism, with the small protein increasing activity of GlpD, an essential enzyme in glycerol catabolism, while the RNA base pairs with and represses galE mRNA, a gene essential for galactose metabolism. The second dual-function RNA studied in this work is Spot 42, a 109-nucleotide RNA known to base pair with and repress mRNAs encoding proteins involved in the metabolism of non-preferred carbon sources. Although Spot 42 is a well-characterized base pairing small RNA (sRNA) in E. coli, this work shows it also encodes a 15-amino acid protein, SpfP. SpfP was found to bind to cAMP receptor protein (CRP) and block activation of some target genes. For both AzuCR and Spot 42, the coding sequence for the small protein overlaps the base pairing region, and we have observed that translation interferes with base pairing activity suggesting competition between the sRNA and mRNA activities. Finally, a synthetic dual-function RNA was constructed from the Escherichia coli sRNA MgrR and the mRNA for the small protein MgtS. Various versions of this hybrid molecule are used to probe how the organization of components is important for the proper functioning of a dual-function RNA. These three studies highlight the complexities of regu