Mara Giangrossi

After a 37 to 10°C temperature downshift the level of translation initiation factor IF2, like that of IF1 and IF3, increases at least 3-fold with respect to the ribosomes. To clarify the mechanisms and conditions leading to cold-stress induction of infB expression, the consequences of this temperature shift on infB (IF2) transcription, infB mRNA stability and translation were analysed. The Escherichia coli gene encoding IF2 is part of the metY-nusA-infB operon that contains three known promoters (P-1, P0 and P2) in addition to two promoters P3 and P4 identified in this study, the latter committed to the synthesis of a monocistronic mRNA encoding exclusively IF2. The results obtained indicate that the increased level of IF2 following cold stress depends on three mechanisms: (i) activation of all the promoters of the operon, P-1 being the most cold-responsive, as a likely consequence of the reduction of the ppGpp level that follows cold stress; (ii) a large increase in infB mRNA half-life and (iii) the cold-shock induced translational bias that ensures efficient translation of infB mRNA by the translational apparatus of cold shocked cells. A comparison of the mechanisms responsible for the cold shock induction of the three initiation factors is also presented.

The icsA gene of Shigella encodes a structural protein involved in colonization of the intestinal mucosa by bacteria. This gene is expressed upon invasion of the host and is controlled by a complex regulatory circuit involving the nucleoid protein H-NS, the AraC-like transcriptional activator VirF, and a 450 nt antisense RNA (RnaG) acting as transcriptional attenuator. We investigated on the interplay of these factors at the molecular level. DNase I footprints reveal that both H-NS and VirF bind to a region including the icsA and RnaG promoters. H-NS is shown to repress icsA transcription at 30°C but not at 37°C, suggesting a significant involvement of this protein in the temperature-regulated expression of icsA. We also demonstrate that VirF directly stimulates icsA transcription and is able to alleviate H-NS repression in vitro. According to these results, icsA expression is derepressed in hns- background and overexpressed when VirF is provided in trans. Moreover, we find that RnaG-mediated transcription attenuation depends on 80 nt at its 5′-end, a stretch carrying the antisense region. Bases engaged in the initial contact leading to sense–antisense pairing have been identified using synthetic RNA and DNA oligonucleotides designed to rebuild and mutagenize the two stem–loop motifs of the antisense region.

The virulence gene icsA of Shigella flexneri encodes an invasion protein crucial for host colonization by pathogenic bacteria. Within the intergenic region virA-icsA, we have discovered a new gene that encodes a non-translated antisense RNA (named RnaG), transcribed in cis on the complementary strand of icsA. In vitro transcription assays show that RnaG promotes premature termination of transcription of icsA mRNA. Transcriptional inhibition is also observed in vivo by monitoring the expression profile in Shigella by real-time polymerase chain reaction and when RnaG is provided in trans. Chemical and enzymatic probing of the leader region of icsA mRNA either free or bound to RnaG indicate that upon hetero-duplex formation an intrinsic terminator, leading to transcription block, is generated on the nascent icsA mRNA. Mutations in the hairpin structure of the proposed terminator impair the RnaG mediated-regulation of icsA transcription. This study represents the first evidence of transcriptional attenuation mechanism caused by a small RNA in Gram-negative bacteria. We also present data on the secondary structure of the antisense region of RnaG. In addition, alternatively silencing icsA and RnaG promoters, we find that transcription from the strong RnaG promoter reduces the activity of the weak convergent icsA promoter through the transcriptional interference regulation.

Expression of Escherichia coli infC, which encodes translation initiation factor IF3 and belongs to a transcriptional unit containing several promoters and terminators, is enhanced after cold shock, causing a transient increase of the IF3/ribosomes ratio. Here we show that after cold shock the two less used promoters (P_T and P_I1) remain active and/or are activated, resulting in de novo infC transcription and IF3 synthesis. These two events are partly responsible for the stoichiometric imbalance of the IF3/ribosomes ratio that contributes to establishing the cold-shock translational bias whereby cold-shock mRNAs are preferentially translated by cold-stressed cells while bulk mRNAs are discriminated against. Analysis of the IF3 functions at low temperature sheds light on the molecular mechanism by which IF3 contributes to the cold-shock translational bias. IF3 was found to cause a strong rate increase of fMet-tRNA binding to ribosomes programmed with cold-shock mRNA, an activity essential for the rapid formation of “30S initiation complexes” at low temperature. The increased IF3/ribosome ratio occurring during cold adaptation was also essential to overcome the higher stability of 70S monomers at low temperature so as to provide a sufficient pool of dissociated 30S subunits capable of “70S initiation complex” formation. Finally, at low temperature IF3 was shown to be endowed with the capacity of discriminating against translation of non-cold-shock mRNAs by a cold-shock-specific “fidelity” function operating with a mechanism different from those previously described, insofar as IF3 does not interfere with formation of 30S initiation complex containing these mRNAs, but induces the formation of nonproductive 70S initiation complexes.

Summary: Escherichia coli infA is transcribed from two promoters, P1 and P2, into a longer and a shorter mRNA encoding translation initiation factor IF1. Although P1 is intrinsically stronger than P2, the shorter half‐life of its transcripts causes the steady‐state level of the P2 transcript to be substantially higher than that of P1 during growth at 37°C. After cold‐shock, de novo transcription and translation of infA contribute to the transient increase of the IF1/ribosomes ratio, which is partially responsible for translational bias consisting in the preferential translation of cold‐shock mRNAs in the cold. Cold‐stress induction of infA expression is mainly due to the high activity of P1 at low temperature, which is further increased by transcriptional stimulation by CspA and by an increased transcript stability. Furthermore, the longer infA mRNA originating from P1 is preferentially translated at low temperature by the translational machinery of cold‐shocked cells. The increased level of IF1 during cold adaptation is essential for overcoming the higher stability of the 70S monomers at low temperature and for providing a sufficient pool of dissociated 30S subunits capable of initiating translation.

Summary: In the human enteropathogenShigellatranscription ofvirF, the primary regulator of the invasion functions, is strictly temperature‐dependent and is antagonistically mediated by H‐NS and FIS, which bind to specific sites on thevirFpromoter. Here we report on the relevance of DNA geometry to the themoregulation ofvirFand demonstrate that thevirFpromoter hosts a major DNA bend halfway between two H‐NS sites. The bent region has been mutagenizedin vitroto mimic temperature‐induced changes of DNA curvature. Functional analysis of curvature mutants and of promoter constructs in which the two H‐NS sites are phased‐out by a half–helix turn reveals that modifying the spatial relationships between these sites severely affects the interaction of H‐NS with thevirFpromoter, as well as itsin vivoandin vitrotemperature‐dependent activity. The role of promoter curvature as thermosensor is also compatible with the present observation that, with increasing temperature, thevirFbending centre moves downstream at a rate having its maximum around the transition temperature, abruptly unmasking a binding site for the transcriptional activator FIS.

Summary: Expression of Escherichia coli hupA and hupB, the structural genes encoding the most abundant nucleoid‐associated proteins HUα and HUβ has been studied during cold shock. This article demonstrates that: (i) transcriptional expression of hupA is blocked following a sudden temperature downshift (from 37°C to 10°C), whereas transcription of hupB from the P2 and P3 promoters is maintained at a constitutive level and is activated de novo from the P4 promoter; (ii) all three hupB mRNAs (transcribed from the three natural promoters P2, P3 and P4) become much more stable than the single hupA transcript; and (iii) the hupB transcripts, unlike that of hupA, are efficiently translated in vivo during cold acclimation and can be actively translated in vitro at low temperature. Taken together, the results indicate that during cold shock the expression of the HUβ subunit is preferentially stimulated and that of HUα repressed, suggesting that an altered HUα to HUβ expression ratio resulting in an increase of HUα/HUβ heterodimers and/or (HUβ)₂ homodimers may play an important role during cold adaptation.