Regulation of heterogenous lexA expression in staphylococcus aureus by an antisense RNA originating from transcriptional read-through upon natural mispairings in the sbrB intrinsic terminator

Bacterial genomes are pervasively transcribed, generating a wide variety of antisense RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the transcription termination process. Previous transcriptome analyses revealed that the lexA gene from Staphylococcus aureus, which encodes the main SOS response regulator, is affected by the presence of an asRNA. Here, we show that the lexA antisense RNA (lexA-asRNA) is generated by a TRE on the intrinsic terminator (TTsbrB) of the sbrB gene, which is located downstream of lexA, in the opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TTsbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TTsbrB prevented lexA-asRNA transcription. The level of lexA-asRNA directly correlated with cellular stress since the expressions of sbrB and lexA-asRNA depend on the stress transcription factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TTsbrB, suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections with downstream genes and, ultimately, transcriptomic variability among S. aureus strains.This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant no. 646869 to A.T.-A.) and by the Spanish Ministry of Science and Innovation grants (BIO2017-83035-R to I.L. and PID2019-105216GB-I00 to A.T.-A.). Funding for open access charge was provided by the CSIC Open Access Publication Support Initiative, Unit of Information Resources for Research (URICI)

Maestro of regulation: Riboswitches orchestrate gene expression at the levels of translation, transcription and mRNA decay

Riboswitches are RNA regulators that control gene expression by modulating their structure in response to metabolite binding. The study of mechanisms by which riboswitches modulate gene expression is crucial to understand how riboswitches are involved in maintaining cellular homeostasis. Previous reports indicate that riboswitches can control gene expression at the level of translation, transcription or mRNA decay. However, there are very few described examples where riboswitches regulate multiple steps in gene expression. Recent studies of a translation-regulating, TPP-dependent riboswitch have revealed that ligand binding is also involved in the control of mRNA levels. In this model, TPP binding to the riboswitch leads to the inhibition of translation, which in turn allows for Rho-dependent transcription termination. Thus, mRNA levels are indirectly controlled through ribosome occupancy. This is in contrast to other riboswitches that directly control mRNA levels by modulating the access of regulatory sequences involved in either Rho-dependent transcription termination or RNase E cleavage activity. Together, these findings indicate that riboswitches modulate both translation initiation and mRNA levels using multiple strategies that direct the outcome of gene expression.Peer reviewe

An antisense RNA connects SOS-response to cellular stress

Trabajo presentado en el 7th Congress of European Microbiologists (FEMS2017), celebrado en Valencia (España), del 9 al 13 de julio de 2017Peer reviewe

Un tránscrito antisentido conecta el estrés celular con la respuesta SOS

Póster presentado en la XI Reunión del Grupo de Microbiología Molecular de la Sociedad Española de Microbiología (SEM), celebrada en Sevilla del 6 al 8 de septiembre de 2016.La determinación precisa de los mapas transcriptómicos bacterianos ha revelado que el número de regiones cromosómicas con transcripción solapante es mucho mayor de lo que habíamos imaginado1. Estos RNAs antisentidos (asRNAs) se pueden generar de dos maneras diferentes: i) por la presencia de un promotor situado en la cadena contraria a una región codificante (CDS) ó ii) por la deficiencia en la parada de transcripción entre genes convergentes. El segundo caso ocurre cuando no existe un terminador de transcripción (TT) entre los genes o la estructura del TT es inestable, generando así regiones 3’ solapantes1,2,3. También, existe la posibilidad de que la formación de la estructura terminadora pueda ser modificada por factores externos tal y como ocurre en algunos riboswitches. Todo ello hace que el nivel de antisentido dependa del promotor de los genes convergentes y, si existe, del TT presente entre ellos. En cualquier caso, la transcripción solapante en las regiones 3’, incrementa la complejidad de la organización de los genes en el cromosoma al establecer interconexiones entre los mismos además de la establecida por los operones. En un trabajo previo, descubrimos que esta transcripción solapante afecta a la expresión de varios reguladores de Staphylococcus aureus. Uno de ellos correspondía al gen lexA, que codifica para el principal regulador de la respuesta SOS1. Mediante Northern blot determinamos que el transcrito antisentido se genera a partir del promotor SigB del small RNA sbrB. Dicho transcrito incluye la CDS de SbrB, una región que solapa completamente con el mRNA de lexA y la CDS de SosA. Para que este RNA de 1,4 Kb se genere, la RNA polimerasa debe evitar el TT de sbrB, finalizando su transcripción en el TT de sosA. El reemplazo del promotor SigB de sbrB por uno constitutivo, provoca cambios en la expresión de LexA cuando la respuesta SOS es inducida por Mitomicina, lo que demuestra la interconexión entre ambas vías de regulación. Aunque son necesarios más experimentos para comprender este mecanismo de regulación, este estudio ejemplifica la complejidad transcritómica presente en las bacterias, donde un mismo transcrito puede contener múltiples elementos funcionales.Agradecimientos: al Ministerio de Economía y Competitividad y al Consejo Europeo de Investigación por la financiación de los proyectos de investigación BFU2014-56698-P y ERC-2014-CoG-646869, respectivamente. A la Universidad Pública de Navarra por la financiación del contrato pre-doctoral de C.C.Peer Reviewe

NtcA Regulates patA Expression in Anabaena sp. Strain PCC 7120▿

patA expression is induced 3 to 6 h after nitrogen step-down. We establish that the transcription of patA is under the positive control of NtcA. The patA promoter region shows two conserved NtcA-binding boxes. These NtcA-binding sites and their interaction with NtcA are key elements for patA expression in heterocysts

An antisense RNA connnects SigB-associated stress wigh SOS-response

Trabajo presentado en el 5th Meeting of Regulation with RNA in Bacteria and Archaea, celebrado en Sevilla (España), del 19 al 22 de marzo de 2018Peer reviewe

Variaciones nucleotidicas en la secuencia de los terminadores de transcripcion generan RNA antisentidos funcionales

Trabajo presentado en la XII Reunión del Grupo Especializado SEM, celebrada en Zaragoza (España) del 5 al 7 de septiembre de 2018Peer reviewe

Comparative study between transcriptionally- and translationally-acting adenine riboswitches reveals key differences in riboswitch regulatory mechanisms

Many bacterial mRNAs are regulated at the transcriptional or translational level by ligand-binding elements called riboswitches. Although they both bind adenine, the adenine riboswitches of Bacillus subtilis and Vibrio vulnificus differ by controlling transcription and translation, respectively. Here, we demonstrate that, beyond the obvious difference in transcriptional and translational modulation, both adenine riboswitches exhibit different ligand binding properties and appear to operate under different regulation regimes (kinetic versus thermodynamic). While the B. subtilis pbuE riboswitch fully depends on co-transcriptional binding of adenine to function, the V. vulnificus add riboswitch can bind to adenine after transcription is completed and still perform translation regulation. Further investigation demonstrates that the rate of transcription is critical for the B. subtilis pbuE riboswitch to perform efficiently, which is in agreement with a cotranscriptional regulation. Our results suggest that the nature of gene regulation control, that is transcription or translation, may have a high importance in riboswitch regulatory mechanisms

Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation.

International audienceOn the basis of nascent transcript sequencing, it has been postulated but never demonstrated that transcriptional pausing at translation start sites is important for gene regulation. Here we show that the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch contains a regulatory pause site in the translation initiation region that acts as a checkpoint for thiC expression. By biochemically probing nascent transcription complexes halted at defined positions, we find a narrow transcriptional window for metabolite binding, in which the downstream boundary is delimited by the checkpoint. We show that transcription complexes at the regulatory pause site favour the formation of a riboswitch intramolecular lock that strongly prevents TPP binding. In contrast, cotranscriptional metabolite binding increases RNA polymerase pausing and induces Rho-dependent transcription termination at the checkpoint. Early transcriptional pausing may provide a general mechanism, whereby transient transcriptional windows directly coordinate the sensing of environmental cues and bacterial mRNA regulation