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Bacterial small RNAs regulate the expression of multiple genes through imperfect base‐pairing with target mRNAs mediated by RNA chaperone proteins such as Hfq. GcvB is the master sRNA regulator of amino acid metabolism and transport in a wide range of Gram‐negative bacteria. Recently, independent RNA‐seq approaches identified a plethora of transcripts interacting with GcvB in Escherichia coli. In this study, the compilation of RIL‐seq, CLASH, and MAPS data sets allowed us to identify GcvB targets with high accuracy. We validated 21 new GcvB targets repressed at the posttranscriptional level, raising the number of direct targets to >50 genes in E. coli. Among its multiple seed sequences, GcvB utilizes either R1 or R3 to regulate most of these targets. Furthermore, we demonstrated that both R1 and R3 seed sequences are required to fully repress the expression of gdhA, cstA, and sucC genes. In contrast, the ilvLXGMEDA polycistronic mRNA is targeted by GcvB through at least four individual binding sites in the mRNA. Finally, we revealed that GcvB is involved in the susceptibility of peptidase‐deficient E. coli strain (Δpeps) to Ala‐Gln dipeptide by regulating both Dpp dipeptide importer and YdeE dipeptide exporter via R1 and R3 seed sequences, respectively. [ABSTRACT FROM AUTHOR]

There is an expanding list of examples by which one mRNA can posttranscriptionally influence the expression of others. This can involve RNA sponges that sequester regulatory RNAs of mRNAs in the same regulon, but the underlying molecular mechanism of such mRNA cross talk remains little understood. Here, we report sponge-mediated mRNA cross talk in the posttranscriptional network of GcvB, a conserved Hfq-dependent small RNA with one of the largest regulons known in bacteria. We show that mRNA decay from the glt IJKL locus encoding an amino acid ABC transporter generates a stable fragment (SroC) that base-pairs with GcvB. This interaction triggers the degradation of GcvB by RNase E, alleviating the GcvB-mediated mRNA repression of other amino acid-related transport and metabolic genes. Intriguingly, since the glt IJKL mRNA itself is a target of GcvB, the SroC sponge seems to enable both an internal feed-forward loop to activate its parental mRNA in cis and activation of many trans-encoded mRNAs in the same pathway. Disabling this mRNA cross talk affects bacterial growth when peptides are the sole carbon and nitrogen sources. [ABSTRACT FROM AUTHOR]