The small regulatory protein Crl binds to σS, the RNA polymerase stationary phase σ factor. Crl facilitates the formation of the σS-associated holoenzyme (EσS) and thereby activates σS-dependent ...genes. Using a real time surface plasmon resonance biosensor, we characterized in greater detail the specificity and mode of action of Crl. Crl specifically forms a 1:1 complex with σS, which results in an increase of the association rate of σS to core RNA polymerase without any effect on the dissociation rate of EσS. Crl is also able to associate with preformed EσS with a higher affinity than with σS alone. Furthermore, even at saturating σS concentrations, Crl significantly increases EσS association with the katN promoter and the productive isomerization of the EσS-katN complex, supporting a direct role of Crl in transcription initiation. Finally, we show that Crl does not bind to σ70 itself but is able at high concentrations to form a weak and transient 1:1 complex with both core RNA polymerase and the σ70-associated holoenzyme, leaving open the possibility that Crl might also exert a side regulatory role in the transcriptional activity of additional non-σS holoenzymes.
The regulation of the Pg promoter, which controls the expression of the meta operon of the 4-hydroxyphenylacetic acid (4-HPA) catabolic pathway ofEscherichia coli W, has been examined through in vivo ...and in vitro experiments. By usingPg-lacZ fusions we have demonstrated that Pg is a promoter only inducible in the stationary phase when cells are grown on glucose as the sole carbon and energy source. This strict catabolite repression control is mediated by the cAMP receptor protein (CRP). This event does not require the presence of the specific HpaR repressor or the 4-HPA permease (HpaX), excluding the involvement of a typical inducer exclusion mechanism. However, the acetic acid excreted in the stationary phase by the cells growing in glucose acts as an overflow metabolite, which can provide the energy to produce cAMP and to adapt the cells rapidly to the utilization of a new less preferred carbon source such as the aromatic compounds. Although Pg is not a ς38-dependent promoter, it is activated by the global regulator integration host factor (IHF) in the stationary phase of growth. Gel retardation assays have demonstrated that both CRP and IHF simultaneously bind to the Pg upstream region. DNase I footprint experiments showed that cAMP-CRP and IHF binding sites are centered at −61.5 and −103, respectively, with respect to the transcription start site +1 of the Pg promoter.
The RpoS/σ(S) sigma subunit of RNA polymerase (RNAP) activates transcription of stationary phase genes in many Gram-negative bacteria and controls adaptive functions, including stress resistance, ...biofilm formation and virulence. In this study, we address an important but poorly understood aspect of σ(S)-dependent control, that of a repressor. Negative regulation by σ(S) has been proposed to result largely from competition between σ(S) and other σ factors for binding to a limited amount of core RNAP (E). To assess whether σ(S) binding to E alone results in significant downregulation of gene expression by other σ factors, we characterized an rpoS mutant of Salmonella enterica serovar Typhimurium producing a σ(S) protein proficient for Eσ(S) complex formation but deficient in promoter DNA binding. Genome expression profiling and physiological assays revealed that this mutant was defective for negative regulation, indicating that gene repression by σ(S) requires its binding to DNA. Although the mechanisms of repression by σ(S) are likely specific to individual genes and environmental conditions, the study of transcription downregulation of the succinate dehydrogenase operon suggests that σ competition at the promoter DNA level plays an important role in gene repression by Eσ(S).
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