Structural basis of σ54 displacement and promoter escape in bacterial transcription.

Gene transcription is a fundamental cellular process carried out by RNA polymerase (RNAP). Transcription initiation is highly regulated, and in bacteria, transcription initiation is mediated by sigma (σ) factors. σ recruits RNAP to the promoter DNA region, located upstream of the transcription start site (TSS) and facilitates open complex formation, where double-stranded DNA is opened up into a transcription bubble and template strand DNA is positioned inside RNAP for initial RNA synthesis. During initial transcription, RNAP remains bound to σ and upstream DNA, presumably with an enlarging transcription bubble. The release of RNAP from upstream DNA is required for promoter escape and processive transcription elongation. Bacteria sigma factors can be broadly separated into two classes with the majority belonging to the σ70 class, represented by the σ70 that regulates housekeeping genes. σ54 forms a class on its own and regulates stress response genes. Extensive studies on σ70 have revealed the molecular mechanisms of the σ70 dependent process while how σ54 transitions from initial transcription to elongation is currently unknown. Here, we present a series of cryo-electron microscopy structures of the RNAP-σ54 initial transcribing complexes with progressively longer RNA, which reveal structural changes that lead to promoter escape. Our data show that initially, the transcription bubble enlarges, DNA strands scrunch, reducing the interactions between σ54 and DNA strands in the transcription bubble. RNA extension and further DNA scrunching help to release RNAP from σ54 and upstream DNA, enabling the transition to elongation

Effect of CRP and its ARI mutant CRP on the p1p2 expression in the double mutant TP2339-1 detected by primer extension analysis

<p><b>Copyright information:</b></p><p>Taken from "Interplay between CRP-cAMP and PII-Ntr systems forms novel regulatory network between carbon metabolism and nitrogen assimilation in "</p><p></p><p>Nucleic Acids Research 2007;35(5):1432-1440.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865078.</p><p>© 2007 The Author(s).</p> Lane 5 is G marker ladder. Cells were grown in M63 minimal medium with glycerol (0.4% w/v) as the sole carbon source, and glutamine (0.2% w/v) as nitrogen source, in the absence/presence of exogenous cAMP (2 mM)

Effect of CRP and its ARI mutant CRP on the uridylylation status of GlnB in the double mutant TP2339-1 and triple mutant BD4000

<p><b>Copyright information:</b></p><p>Taken from "Interplay between CRP-cAMP and PII-Ntr systems forms novel regulatory network between carbon metabolism and nitrogen assimilation in "</p><p></p><p>Nucleic Acids Research 2007;35(5):1432-1440.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865078.</p><p>© 2007 The Author(s).</p> All strains (lanes 1–8) were grown in M63 minimal medium with glycerol (0.4% w/v) as the sole carbon source, and glutamine (0.2% w/v) as nitrogen source, in the absence/presence of exogenous cAMP (2 mM). TP2339-1 was grown in nitrogen-sufficient medium with 40 mM ammonium as control (lane 9)

DNase I footprints of Eσ in the presence or absence of CRP and CRP on p1 (non template strand)

<p><b>Copyright information:</b></p><p>Taken from "Interplay between CRP-cAMP and PII-Ntr systems forms novel regulatory network between carbon metabolism and nitrogen assimilation in "</p><p></p><p>Nucleic Acids Research 2007;35(5):1432-1440.</p><p>Published online 6 Feb 2007</p><p>PMCID:PMC1865078.</p><p>© 2007 The Author(s).</p> () Titration with increasing concentrations of CRP (lanes 2 and 7, 30 nM; lanes 3 and 8, 100 nM; lanes 4 and 9, 300 nM) were performed in the absence (2–4) or presence of 50 nM Eσ (lanes 7–9). Lane 11 is A + G marker ladder. The protected regions were monitored by adding increasing concentrations of CRP in the presence or absence of Eσ. () Titration with increasing concentrations of CRP (lane 3, 33 nM; lane 4, 100 nM; lane 5, 300 nM) and CRP (lane 10, 33 nM; lane 11, 100 nM; lane 12, 300 nM) were performed in the presence of 25 nM Eσ (lanes 3–5 and 10–12). Lane 7 is A + G marker ladder. The limits of protected regions are indicated. Note that wild-type CRP is recruited to a site centred at −61.5 by Eσ-RNA polymerase, which suggests a higher affinity for DNA binding of Eσ than that of CRP

Proteomic/metabolic data and mathematical model of nitrogen assimilation

Experimental data and mathematical model accompanying the paper 'GlnK facilitates the dynamic regulation of bacterial nitrogen assimilation' by Gosztolai et al. (2017) Biophys J. <div><br></div><div>Data.xlsx contains concurrent <i>in vivo</i> time-courses of metabolite, total protein and PTM protein concentrations in response to time-varying external ammonium levels. </div><div><br></div><div>Model.txt contains the mathematical model in the paper in SBML language.</div