Riboswitches and attenuators are cis-regulatory RNA elements, most of which control bacterial gene expression via metabolite-mediated, premature transcription termination. We developed an unbiased experimental approach for genome-wide discovery of such ribo-regulators in bacteria. We also devised an experimental platform that quantitatively measures the in-vivo activity of all such regulators in parallel, and enables rapid screening for ribo-regulators that respond to metabolites of choice. Using this approach we detected numerous antibiotic-responsive riboregulators that control antibiotic resistance genes in pathogens and in the human microbiome. Studying one such regulator in Listeria monocytogenes revealed an attenuation mechanism mediated by antibiotic-stalled ribosomes. Our results expose broad roles for conditional termination in regulating antibiotic resistance, and provide a tool for discovering riboswitches and attenuators that respond to novel ligands.Riboswitches and attenuators are 5'UTR-residing, cis-regulatory RNA elements (riboregulators) that tune gene expression in bacteria by sensing key metabolites, amino acids, nucleotides and ions (1-6). These RNA elements can regulate the expression of the downstream gene either at the transcription or the translation level. When riboswitches and attenuators control transcription they usually generate a condition-specific, regulated transcriptional terminator, such that termination results in a prematurely aborted transcript whereas read-through generates a full length, productive mRNA (5) (Fig. 1A). In the case of riboswitches, the 5'UTR RNA sensor differentially folds to form a terminator or an antiterminator in the presence or absence of a regulating metabolite, respectively; in attenuators, the formation of a transcriptional terminator is mediated by the rate of translation of an upstream ORF (uORF), as exemplified in the Trp operon (4). Regulation by conditional termination controls key processes in bacteria including core metabolism (7,8), motility (9) biofilm formation (9, 10), and virulence (11,12). Riboswitches enable optimization of metabolite production in bacterial expression systems (13,14), are readily *
Transcription termination in bacteria can occur either via Rho-dependent or independent (intrinsic) mechanisms. Intrinsic terminators are composed of a stem-loop RNA structure followed by a uridine stretch and are known to terminate in a precise manner. In contrast, Rho-dependent terminators have more loosely defined characteristics and are thought to terminate in a diffuse manner. While transcripts ending in an intrinsic terminator are protected from 3′-5′ exonuclease digestion due to the stem-loop structure of the terminator, it remains unclear what protects Rho-dependent transcripts from being degraded. In this study, we mapped the exact steady-state RNA 3′ ends of hundreds of Escherichia coli genes terminated either by Rho-dependent or independent mechanisms. We found that transcripts generated from Rho-dependent termination have precise 3′-ends at steady state. These termini were localized immediately downstream of energetically stable stem-loop structures, which were not followed by uridine rich sequences. We provide evidence that these structures protect Rho-dependent transcripts from 3′-5′ exonucleases such as PNPase and RNase II, and present data localizing the Rho-utilization (rut) sites immediately downstream of these protective structures. This study represents the first extensive in-vivo map of exact RNA 3′-ends of Rho-dependent transcripts in E. coli.
Riboswitches are ligand-binding elements contained within the 5' untranslated regions of bacterial transcripts, which generally regulate expression of downstream open reading frames. Here, we show that in Listeria monocytogenes, a riboswitch that binds vitamin B12 controls expression of a noncoding regulatory RNA, Rli55. Rli55, in turn, controls expression of the eut genes, whose products enable ethanolamine utilization and require B12 as a cofactor. Defects in ethanolamine utilization, or in its regulation by Rli55, significantly attenuate Listeria virulence in mice. Rli55 functions by sequestering the two-component response regulator EutV by means of a EutV-binding site contained within the RNA. Thus, Rli55 is a riboswitch-regulated member of the small group of regulatory RNAs that function by sequestering a protein and reveals a distinctive mechanism of signal integration in bacterial gene regulation.
Bacterial operons synchronize the expression of multiple genes by placing them under the control of a shared promoter. It was previously shown that polycistronic transcripts can undergo differential RNA decay, leaving some genes within the polycistron more stable than others, but the extent of regulation by differential mRNA decay or its evolutionary conservation remains unknown. Here, we find that a substantial fraction of E. coli genes display non-uniform mRNA stoichiometries despite being coded from the same operon. We further show that these altered operon stoichiometries are shaped post-transcriptionally by differential mRNA decay, which is regulated by RNA structures that protect specific regions in the transcript from degradation. These protective RNA structures are generally coded within the protein-coding regions of the regulated genes and are frequently evolutionarily conserved. Furthermore, we provide evidence that differences in ribosome densities across polycistronic transcript segments, together with the conserved structural RNA elements, play a major role in the differential decay process. Our results highlight a major role for differential mRNA decay in shaping bacterial transcriptomes.
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