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After an RNA polymerase reaches a terminator, instead of dissociating from the template, it may diffuse along the DNA and recommence RNA synthesis from the previous or a different promoter. Magnetic tweezers were used to monitor such secondary transcription and determine the effects of low forces assisting or opposing translocation, protein roadblocks, and transcription factors. Remarkably, up to 50% of Escherichia coli (E. coli) RNA polymerases diffused along the DNA after termination. Force biased the direction of diffusion (sliding) and the velocity increased rapidly with force up to 0.7 pN and much more slowly thereafter. Sigma factor 70 (σ 70 ) likely remained associated with the DNA promoting sliding and enabling re-initiation from promoters in either orientation. However, deletions of the α-C-terminal domains severely limited the ability of RNAP to turn around between successive rounds of transcription. The addition of elongation factor NusG, which competes with σ 70 for binding to RNAP, limited additional rounds of transcription. Surprisingly, sliding RNA polymerases blocked by a DNA-bound lac repressor could slowly re-initiate transcription and were not affected by NusG, suggesting a σ-independent pathway. Low forces effectively biased promoter selection suggesting a prominent role for topological entanglements that affect RNA polymerase translocation., (© 2024. The Author(s).)

Clostridium thermocellum is a potential microbial platform to convert abundant plant biomass to biofuels and other renewable chemicals. It efficiently degrades lignocellulosic biomass using a surface displayed cellulosome, a megadalton sized multienzyme containing complex. The enzymatic composition and architecture of the cellulosome is controlled by several transmembrane biomass-sensing RsgI-type anti-σ factors. Recent studies suggest that these factors transduce signals from the cell surface via a conserved RsgI extracellular (CRE) domain (also called a periplasmic domain) that undergoes autoproteolysis through an incompletely understood mechanism. Here we report the structure of the autoproteolyzed CRE domain from the C. thermocellum RsgI9 anti-σ factor, revealing that the cleaved fragments forming this domain associate to form a stable α/β/α sandwich fold. Based on AlphaFold2 modeling, molecular dynamics simulations, and tandem mass spectrometry, we propose that a conserved Asn-Pro bond in RsgI9 autoproteolyzes via a succinimide intermediate whose formation is promoted by a conserved hydrogen bond network holding the scissile peptide bond in a strained conformation. As other RsgI anti-σ factors share sequence homology to RsgI9, they likely autoproteolyze through a similar mechanism., (© 2024 Wiley Periodicals LLC.)

Leptospirosis is a widespread zoonotic infectious disease of human and veterinary concern caused by pathogenic spirochetes of the genus Leptospira. To date, little progress towards understanding leptospiral pathogenesis and identification of virulence factors has been made, which is the main bottleneck for developing effective measures against the disease. Some leptospiral proteins, including LipL32, Lig proteins, LipL45, and LipL21, are being considered as potential virulence factors or vaccine candidates. However, their function remains to be established. LipL45 is the most expressed membrane lipoprotein in leptospires, upregulated when the bacteria are transferred to temperatures resembling the host, expressed during infection, suppressed after culture attenuation, and known to suffer processing in vivo and in vitro, generating fragments. Based on body of evidence, we hypothesized that the LipL45 processing might occur by an auto-cleavage event, deriving two fragments. The results presented here, based on bioinformatics, structure modeling analysis, and experimental data, corroborate that LipL45 processing probably includes a self-catalyzed non-proteolytic event and suggest the participation of LipL45 in cell-surface signaling pathways, as the protein shares structural similarities with bacterial sigma regulators. Our data indicate that LipL45 might play an important role in response to environmental conditions, with possible function in the adaptation to the host., Competing Interests: Declaration of competing interest The authors have no conflicts to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bacterial RNAP needs to form holoenzyme with σ factors to initiate transcription. While Staphylococcus aureus σ A controls housekeeping functions, S. aureus σ B regulates virulence, biofilm formation, persistence, cell internalization, membrane transport, and antimicrobial resistance. Besides the sequence difference, the spacers between the -35 element and -10 element of σ B regulated promoters are shorter than those of σ A regulated promoters. Therefore, how σ B recognizes and initiates transcription from target promoters can not be inferred from that of the well studied σ. Here, we report the cryo-EM structures of S. aureus RNAP-promoter open complexes comprising σ A and σ B , respectively. Structural analyses, in combination with biochemical experiments, reveal the structural basis for the promoter specificity of S. aureus transcription. Although the -10 element of σ A regulated promoters is recognized by domain σ A 2 as single-stranded DNA, the -10 element of σ B regulated promoters is co-recognized by domains σ B 2 and σ B 3 as double-stranded DNA, accounting for the short spacers of σ B regulated promoters. S. aureus RNAP is a validated target of antibiotics, and our structures pave the way for rational drug design targeting S. aureus RNAP., (© 2024. The Author(s).)

During the past few decades, a wealth of knowledge has been made available for the transcription machinery in bacteria from the structural, functional and mechanistic point of view. However, comparatively little is known about the homooligomerization of the multisubunit M. tuberculosis RNA polymerase (RNAP) enzyme and its functional relevance. While E. coli RNAP has been extensively studied, many aspects of RNAP of the deadly pathogenic M. tuberculosis are still unclear. We used biophysical and biochemical methods to study the oligomerization states of the core and holoenzymes of M. tuberculosis RNAP. By size exclusion chromatography and negative staining Transmission Electron Microscopy (TEM) studies and quantitative analysis of the TEM images, we demonstrate that the in vivo reconstituted RNAP core enzyme (α2ββ'ω) can also exist as dimers in vitro. Using similar methods, we also show that the holoenzyme (core + σ A ) does not dimerize in vitro and exist mostly as monomers. It is tempting to suggest that the oligomeric changes that we see in presence of σ A factor might have functional relevance in the cellular process. Although reported previously in E. coli, to our knowledge we report here for the first time the study of oligomeric nature of M. tuberculosis RNAP in presence and absence of σ A factor., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Gene expression in Escherichia coli is controlled by well-established mechanisms that activate or repress transcription. Here, we identify CedA as an unconventional transcription factor specifically associated with the RNA polymerase (RNAP) σ 70 holoenzyme. Structural and biochemical analysis of CedA bound to RNAP reveal that it bridges distant domains of β and σ 70 subunits to stabilize an open-promoter complex. CedA does so without contacting DNA. We further show that cedA is strongly induced in response to amino acid starvation, oxidative stress and aminoglycosides. CedA provides a basal level of tolerance to these clinically relevant antibiotics, as well as to rifampicin and peroxide. Finally, we show that CedA modulates transcription of hundreds of bacterial genes, which explains its pleotropic effect on cell physiology and pathogenesis., (© 2024. The Author(s).)

Transcription of specific genes in bacteria under environmental stress is frequently initiated by extracytoplasmic function (ECF) σ factors. ECFs σ factors harbour two conserved domains, σ 2 and σ 4 , for transcription initiation by recognition of the promoter region and recruitment of RNA polymerase (RNAP). The crystal structure of Streptomyces tsukubaensis SigG1, an ECF56-family σ factor, was determined revealing σ 2 , σ 4 and the additional carboxi-terminal domain SnoaL_2 tightly packed in a compact conformation. The structure of anti-sigma RsfG was also determined by X-ray crystallography and shows a rare β-barrel fold. Analysis of the metal binding motifs inside the protein barrel are consistent with Fe(III) binding, which is in agreement with previous findings that the Streptomyces tsukubaensis ECF56 SigG1-RsfG system is involved in metal-ion homeostasis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In enterobacteria such as Escherichia coli, the general stress response is mediated by σ s , the stationary phase dissociable promoter specificity subunit of RNA polymerase. σ s is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which is thought to be stimulated by the phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of full-length RssB bound to a beryllofluoride phosphomimic. Compared to the structure of RssB bound to the IraD anti-adaptor, our new RssB structure with bound beryllofluoride reveals conformational differences and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the interdomain segmented helical linker. These are accompanied by masking of the α4-β5-α5 (4-5-5) "signaling" face of the RssB receiver domain by its C-terminal domain. Critically, using hydrogen-deuterium exchange mass spectrometry, we identify σ s -binding determinants on the 4-5-5 face, implying that this surface needs to be unmasked to effect an interdomain interface switch and enable full σ s engagement and hand-off to ClpXP. In activated receiver domains, the 4-5-5 face is often the locus of intermolecular interactions, but its masking by intramolecular contacts upon phosphorylation is unusual, emphasizing that RssB is a response regulator that undergoes atypical regulation., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Autoproteolysis has been discovered to play key roles in various biological processes, but functional autoproteolysis has been rarely reported for transmembrane signaling in prokaryotes. In this study, an autoproteolytic effect was discovered in the conserved periplasmic domain of anti-σ factor RsgIs from Clostridium thermocellum , which was found to transmit extracellular polysaccharide-sensing signals into cells for regulation of the cellulosome system, a polysaccharide-degrading multienzyme complex. Crystal and NMR structures of periplasmic domains from three RsgIs demonstrated that they are different from all known proteins that undergo autoproteolysis. The RsgI-based autocleavage site was located at a conserved Asn-Pro motif between the β1 and β2 strands in the periplasmic domain. This cleavage was demonstrated to be essential for subsequent regulated intramembrane proteolysis to activate the cognate SigI, in a manner similar to that of autoproteolysis-dependent activation of eukaryotic adhesion G protein-coupled receptors. These results indicate the presence of a unique prevalent type of autoproteolytic phenomenon in bacteria for signal transduction.

Mycobacterium tuberculosis (Mtb) WhiB3 is an iron-sulfur cluster-containing transcription factor belonging to a subclass of the WhiB-Like (Wbl) family that is widely distributed in the phylum Actinobacteria. WhiB3 plays a crucial role in the survival and pathogenesis of Mtb. It binds to the conserved region 4 of the principal sigma factor (σ A 4 ) in the RNA polymerase holoenzyme to regulate gene expression like other known Wbl proteins in Mtb. However, the structural basis of how WhiB3 coordinates with σ A 4 to bind DNA and regulate transcription is unclear. Here we determined crystal structures of the WhiB3:σ A 4 complex without and with DNA at 1.5 Å and 2.45 Å, respectively, to elucidate how WhiB3 interacts with DNA to regulate gene expression. These structures reveal that the WhiB3:σ A 4 complex shares a molecular interface similar to other structurally characterized Wbl proteins and also possesses a subclass-specific Arg-rich DNA-binding motif. We demonstrate that this newly defined Arg-rich motif is required for WhiB3 binding to DNA in vitro and transcriptional regulation in Mycobacterium smegmatis. Together, our study provides empirical evidence of how WhiB3 regulates gene expression in Mtb by partnering with σ A 4 and engaging with DNA via the subclass-specific structural motif, distinct from the modes of DNA interaction by WhiB1 and WhiB7., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Multisubunit RNA polymerases (RNAPs) associate with initiation factors (σ in bacteria) to start transcription. The σ factors are responsible for recognizing and unwinding promoter DNA in all bacterial RNAPs. Here, we report two cryo-EM structures of cyanobacterial transcription initiation complexes at near-atomic resolutions. The structures show that cyanobacterial RNAP forms an "SI3-σ" arch interaction between domain 2 of σ A (σ 2 ) and sequence insertion 3 (SI3) in the mobile catalytic domain Trigger Loop (TL). The "SI3-σ" arch facilitates transcription initiation from promoters of different classes through sealing the main cleft and thereby stabilizing the RNAP-promoter DNA open complex. Disruption of the "SI3-σ" arch disturbs cyanobacteria growth and stress response. Our study reports the structure of cyanobacterial RNAP and a unique mechanism for its transcription initiation. Our data suggest functional plasticity of SI3 and provide the foundation for further research into cyanobacterial and chloroplast transcription.

Bacterial transcription initiation requires σ factors for nucleation of the transcription bubble. The canonical housekeeping σ factor, σ 70 , nucleates DNA melting via recognition of conserved bases of the promoter -10 motif, which are unstacked and captured in pockets of σ 70 . By contrast, the mechanism of transcription bubble nucleation and formation during the unrelated σ N -mediated transcription initiation is poorly understood. Herein, we combine structural and biochemical approaches to establish that σ N , like σ 70 , captures a flipped, unstacked base in a pocket formed between its N-terminal region I (RI) and extra-long helix features. Strikingly, RI inserts into the nascent bubble to stabilize the nucleated bubble prior to engagement of the obligate ATPase activator. Our data suggest a general paradigm of transcription initiation that requires σ factors to nucleate an early melted intermediate prior to productive RNA synthesis.

Stable 37 °C open complexes (OC) of E. coli RNA polymerase (RNAP) at λP R and T7A1 promoters form at similar rates but have very different lifetimes. To understand the downstream interactions responsible for OC lifetime, how promoter sequence directs them and when they form, we report lifetimes of stable OC and unstable late (I 2 ) intermediates for promoters with different combinations of λP R (L) and T7A1 (T) discriminators, core promoters and UP elements. I 2 lifetimes are similarly short, while stable OC lifetimes differ greatly, determined largely by the discriminator and modulated by core-promoter and UP elements. The free energy change ΔG 3 o for I 2  → stable OC is approximately -4 kcal more favorable for L-discriminator than for T-discriminator promoters. Downstream-truncation at +6 (DT+6) greatly destabilizes OC at L-discriminator but not T-discriminator promoters, making all ΔG 3 o values similar (approximately -4 kcal). Urea reduces OC lifetime greatly by affecting ΔG 3 o . We deduce that urea acts by disfavoring coupled folding of key elements of the β'-clamp, that I 2 is an open-clamp OC, and that clamp-closing in I 2  → stable OC involves coupled folding. Differences in ΔG 3 o between downstream-truncated and full-length promoters yield contributions to ΔG 3 o from interactions with downstream mobile elements (DME) including β-lobe and β'-jaw, more favorable for L-discriminator than for T-discriminator promoters. We deduce how competition between far-downstream DNA and σ 70 region 1.1 affects ΔG 3 o values. We discuss variant-specific ΔG 3 o contributions in terms of the allosteric network by which differences in discriminator and -10 sequence are sensed and transmitted downstream to affect DME-duplex interactions in I 2  → stable OC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In Azospirillum brasilense, an extra-cytoplasmic function σ factor (RpoE10) shows the characteristic 119 amino acid long C-terminal extension found in ECF41-type σ factors, which possesses three conserved motifs (WLPEP, DGGGR, and NPDKV), one in the linker region between the σ 2 and σ 4 , and the other two in the SnoaL_2 domain of the C-terminal extension. Here, we have described the role of the two conserved motifs in the SnoaL_2 domain of RpoE10 in the inhibition and activation of its activity, respectively. Truncation of the distal part of the C-terminal sequence of the RpoE10 (including NPDKV but excluding the DGGGR motif) results in its promoter's activation suggesting autoregulation. Further truncation of the C-terminal sequence up to its proximal part, including NPDKV and DGGGR motif, abolished promoter activation. Replacement of NPDKV motif with NAAAV in RpoE10 increased its ability to activate its promoter, whereas replacement of DGGGR motif led to reduced promoter activation. We have explored the dynamic modulation of σ 2 -σ 4 domains and the relevant molecular interactions mediated by the two conserved motifs of the SnoaL2 domain using molecular dynamics simulation. The analysis enabled us to explain that the NPDKV motif located distally in the C-terminus negatively impacts transcriptional activation. In contrast, the DGGGR motif found proximally of the C-terminal extension is required to activate RpoE10., (© 2022 Wiley Periodicals LLC.)

FliA (also known as σ 28 ), a member of the bacterial σ 70 family of transcription factors, directs RNA polymerase to flagellar late (class 3) promoters and initiates transcription. FliA has been studied in several bacteria, yet its role in spirochetes has not been established. In this report, we identify and functionally characterize a FliA homolog (TDE2683) in the oral spirochete Treponema denticola. Computational, genetic, and biochemical analyses demonstrated that TDE2683 has a structure similar to that of the σ 28 of Escherichia coli, binds to σ 28 -dependent promoters, and can functionally replace the σ 28 of E. coli. However, unlike its counterparts from other bacteria, TDE2683 cannot be deleted, suggesting its essential role in the survival of T. denticola. In vitro site-directed mutagenesis revealed that E221 and V231, two conserved residues in the σ 4 region of σ 28 , are indispensable for the binding activity of TDE2683 to the σ 28 -dependent promoter. We then mutated these two residues in T. denticola and found that the mutations impair the expression of flagellin and chemotaxis genes and bacterial motility as well. Cryo-electron tomography analysis further revealed that the mutations disrupt the flagellar symmetry (i.e., number and placement) of T. denticola. Collectively, these results indicate that TDE2683 is a σ 28 transcription factor that regulates the class 3 gene expression and controls the flagellar symmetry of T. denticola. To the best of our knowledge, this is the first report establishing the functionality of FliA in spirochetes. IMPORTANCE Spirochetes are a group of medically important but understudied bacteria. One of the unique aspects of spirochetes is that they have periplasmic flagella (PF, also known as endoflagella) which give rise to their unique spiral shape and distinct swimming behaviors and play a critical role in the pathophysiology of spirochetes. PF are structurally similar to external flagella, but the underpinning mechanism that regulates PF biosynthesis and assembly remains largely unknown. By using the oral spirochete Treponema denticola as a model, this report provides several lines of evidence that FliA, a σ 28 transcriptional factor, regulates the late flagellin gene (class 3) expression, PF assembly, and flagellar symmetry as well, which provides insights into flagellar regulation and opens an avenue to investigate the role of σ 28 in spirochetes.

Alternate sigma factors play a major role in the survival of pathogenic bacteria such as Streptococcus pyogenes in adverse environment conditions. Stress induced sigma factors mediate gene expression under conditions of pathogenesis, dormancy and unusual environmental cues. In the present work, ComX, an alternate sigma factor from S. pyogenes has been characterized. The structures of ComX, RpoB β subunit and RpoC β' subunit of RNA polymerase have been predicted using comparative and homology modelling respectively and validated. Attempts have been made to study RpoB-RpoC-ComX complex interactions with Double Strand (DS) and Single Strand (SS) promoter regions. Stability of these complexes and the promoter melting mechanism have been analysed using Molecular Dynamic (MD) simulations. This study suggests that ComX, although identifies promoter analogous to the alternate sigma factor SigH of M. tuberculosis , follows a distinctive promoter flip out mechanism.Communicated by Ramaswamy H. Sarma.

Clostridium thermocellum is actively being developed as a microbial platform to produce biofuels and chemicals from renewable plant biomass. An attractive feature of this bacterium is its ability to efficiently degrade lignocellulose using surface-displayed cellulosomes, large multi-protein complexes that house different types of cellulase enzymes. Clostridium thermocellum tailors the enzyme composition of its cellulosome using nine membrane-embedded anti-σ factors (RsgI1-9), which are thought to sense different types of extracellular carbohydrates and then elicit distinct gene expression programs via cytoplasmic σ factors. Here we show that the RsgI9 anti-σ factor interacts with cellulose via a C-terminal bi-domain unit. A 2.0 Å crystal structure reveals that the unit is constructed from S1C peptidase and NTF2-like protein domains that contain a potential binding site for cellulose. Small-angle X-ray scattering experiments of the intact ectodomain indicate that it adopts a bi-lobed, elongated conformation. In the structure, a conserved RsgI extracellular (CRE) domain is connected to the bi-domain via a proline-rich linker, which is expected to project the carbohydrate-binding unit ~160 Å from the cell surface. The CRE and proline-rich elements are conserved in several other C. thermocellum anti-σ factors, suggesting that they will also form extended structures that sense carbohydrates., (© 2022 Wiley Periodicals LLC.)

Spx is a global transcriptional regulator in Gram-positive bacteria and has been inferred to efficiently activate transcription upon oxidative stress by engaging RNA polymerase (RNAP) and promoter DNA. However, the precise mechanism by which it interacts with RNAP and promoter DNA to initiate transcription remains obscure. Here, we report the cryo-EM structure of an intact Spx-dependent transcription activation complex (Spx-TAC) from Bacillus subtilis at 4.2 Å resolution. The structure traps Spx in an active conformation and defines key interactions accounting for Spx-dependent transcription activation. Strikingly, an oxidized Spx monomer engages RNAP by simultaneously interacting with the C-terminal domain of RNAP alpha subunit (αCTD) and σA. The interface between Spx and αCTD is distinct from those previously reported activators, indicating αCTD as a multiple target for the interaction between RNAP and various transcription activators. Notably, Spx specifically wraps the conserved -44 element of promoter DNA, thereby stabilizing Spx-TAC. Besides, Spx interacts extensively with σA through three different interfaces and promotes Spx-dependent transcription activation. Together, our structural and biochemical results provide a novel mechanistic framework for the regulation of bacterial transcription activation and shed new light on the physiological roles of the global Spx-family transcription factors., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In the model organism Escherichia coli and related species, the general stress response relies on tight regulation of the intracellular levels of the promoter specificity subunit RpoS. RpoS turnover is exclusively dependent on RssB, a two-domain response regulator that functions as an adaptor that delivers RpoS to ClpXP for proteolysis. Here, we report crystal structures of the receiver domain of RssB both in its unphosphorylated form and bound to the phosphomimic BeF 3 - . Surprisingly, we find only modest differences between these two structures, suggesting that truncating RssB may partially activate the receiver domain to a "meta-active" state. Our structural and sequence analysis points to RssB proteins not conforming to either the Y-T coupling scheme for signaling seen in prototypical response regulators, such as CheY, or to the signaling model of the less understood FATGUY proteins., (© 2021 The Protein Society.)

Bacterial sigma (σ) factor, along with RNA polymerase core enzyme, initiates gene transcription from specific promoter regions and therefore regulates clusters of genes in response to a particular situation. The extracytoplasmic function (ECF) σ factors are a class of alternative σ factors that are often associated with environmental signal transduction across the bacterial membrane, in which external signal triggers the release of active σ from the membrane-anchored anti-σ factor. Gram-positive model organism Bacillus subtilis (B. subtilis) has seven ECF σ factors: σ M , σ V , σ X , σ W , σ Y , σ Z and σ YlaC . Although all these ECF σ factors were found to be involved in B. subtilis antibiotic resistance, σ W is among the most studied and considered to play a pivotal role in responding to antimicrobial stresses. σ W is under tight control and remains deactivated until exposure to external stimuli, after which proteases PrsW and RasP cleave the specific anti-sigma factor-RsiW to release and activate σ W . Membrane anchored protein YsdB is a negative regulator of this activation, possibly via its direct interaction with PrsW and/or RsiW. Importantly, YsdB is well conserved among Bacilli, including pathogenic bacteria like Bacillus cereus. In this study, we describe the chemical shift assignments of the cytoplasmic domain of YsdB (29-130) of B. subtilis in solution as a basis for further interaction studies and structure determination. The near-complete assignment and the solution structure that will follow could provide a further understanding in σ W regulation.

Transcription activation of bacteriophage T4 late genes is accomplished by a transcription activation complex containing RNA polymerase (RNAP), the promoter specificity factor gp55, the coactivator gp33, and a universal component of cellular DNA replication, the sliding clamp gp45. Although genetic and biochemical studies have elucidated many aspects of T4 late gene transcription, no precise structure of the transcription machinery in the process is available. Here, we report the cryo-EM structures of a gp55-dependent RNAP-promoter open complex and an intact gp45-dependent transcription activation complex. The structures reveal the interactions between gp55 and the promoter DNA that mediate the recognition of T4 late promoters. In addition to the σR2 homology domain, gp55 has a helix-loop-helix motif that chaperons the template-strand single-stranded DNA of the transcription bubble. Gp33 contacts both RNAP and the upstream double-stranded DNA. Gp45 encircles the DNA and tethers RNAP to it, supporting the idea that gp45 switches the promoter search from three-dimensional diffusion mode to one-dimensional scanning mode.

Regulation of bacterial stress responding σ S is a sophisticated process and mediated by multiple interacting partners. Controlled proteolysis of σ S is regulated by RssB which maintains minimal level of σ S during exponential growth but then elevates σ S level while facing stresses. Bacteria developed different strategies to regulate activity of RssB, including phosphorylation of itself and production of anti-adaptors. However, the function of phosphorylation is controversial and the mechanism of anti-adaptors preventing RssB-σ S interaction remains elusive. Here, we demonstrated the impact of phosphorylation on the activity of RssB and built the RssB-σ S complex model. Importantly, we showed that the phosphorylation site - D58 is at the interface of RssB-σ S complex. Hence, mutation or phosphorylation of D58 would weaken the interaction of RssB with σ S . We found that the anti-adaptor protein IraD has higher affinity than σ S to RssB and its binding interface on RssB overlaps with that for σ S . And IraD-RssB complex is preferred over RssB-σ S in solution, regardless of the phosphorylation state of RssB. Our study suggests that RssB possesses a two-tier mechanism for regulating σ S . First, phosphorylation of RssB provides a moderate and reversible tempering of its activity, followed by a specific and robust inhibition via the anti-adaptor interaction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ribosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. Here, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ 70 holoenzyme during rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element using dimerized α subunit carboxyl-terminal domains and scrunches the template DNA with the σ finger and β' lid to select the transcription start site favorable for rapid promoter escape. Promoter binding induces conformational change of σ domain 2 that opens a gate for DNA loading and ejects σ 1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding also opens the DNA loading gate, which is not coupled to σ 1.1 ejection and impedes open complex formation. These results provide a molecular basis for the exceptionally active rRNA transcription and its vulnerability to DksA/ppGpp.

σ B , an alternative sigma factor, is usually employed to tackle the general stress response in Staphylococcus aureus and other Gram-positive bacteria. This protein, involved in S. aureus -mediated pathogenesis, is typically blocked by RsbW, an antisigma factor having serine kinase activity. σ B , a σ 70 -like sigma factor, harbors three conserved domains designated σ B2 , σ B3 , and σ B4 . To better understand the interaction between RsbW and σ B or its domains, we have studied their recombinant forms, rRsbW, rσ B , rσ B2 , rσ B3 , and rσ B4 , using different probes. The results show that none of the rσ B domains, unlike rσ B , showed binding to a cognate DNA in the presence of a core RNA polymerase. However, both rσ B2 and rσ B3 , like rσ B , interacted with rRsbW, and the order of their rRsbW binding affinity looks like rσ B > rσ B3 > rσ B2 . Furthermore, the reaction between rRsbW and rσ B or rσ B3 was exothermic and occurred spontaneously. rRsbW and rσ B3 also associate with each other at a stoichiometry of 2:1, and different types of noncovalent bonds might be responsible for their interaction. A structural model of the RsbW-σ B3 complex that has supported our experimental results indicated the binding of rσ B3 at the putative dimeric interface of RsbW. A genetic study shows that the tentative dimer-forming region of RsbW is crucial for preserving its rσ B binding ability, serine kinase activity, and dimerization ability. Additionally, a urea-induced equilibrium unfolding study indicated a notable thermodynamic stabilization of σ B3 in the presence of RsbW. Possible implications of the stabilization data in drug discovery were discussed at length.

In bacteria, rapid adaptation to changing environmental conditions depends on the interplay between housekeeping and alternative σ factors, responsible for transcription of specific regulons by RNA polymerase (RNAP). In comparison with alternative σ factors, primary σs contain poorly conserved region 1.1, whose functions in transcription are only partially understood. We found that a single mutation in region 1.1 in Escherichia coli σ70 rewires transcription regulation during cell growth resulting in profound phenotypic changes. Despite its destabilizing effect on promoter complexes, this mutation increases the activity of rRNA promoters and also decreases RNAP sensitivity to the major regulator of stringent response DksA. Using total RNA sequencing combined with single-cell analysis of gene expression we showed that changes in region 1.1 disrupt the balance between the "greed" and "fear" strategies thus making the cells more susceptible to environmental threats and antibiotics. Our results reveal an unexpected role of σ region 1.1 in growth-dependent transcription regulation and suggest that changes in this region may facilitate rapid switching of RNAP properties in evolving bacterial populations., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacterial RNA polymerase is a potent target for antibiotics, which utilize a plethora of different modes of action, some of which are still not fully understood. Ureidothiophene (Urd) was found in a screen of a library of chemical compounds for ability to inhibit bacterial transcription. The mechanism of Urd action is not known. Here, we show that Urd inhibits transcription at the early stage of closed complex formation by blocking interaction of RNA polymerase with the promoter -10 element, while not affecting interactions with -35 element or steps of transcription after promoter closed complex formation. We show that mutation in the region 1.2 of initiation factor σ decreases sensitivity to Urd. The results suggest that Urd may directly target σ region 1.2, which allosterically controls the recognition of -10 element by σ region 2. Alternatively, Urd may block conformational changes of the holoenzyme required for engagement with -10 promoter element, although by a mechanism distinct from that of antibiotic fidaxomycin (lipiarmycin). The results suggest a new mode of transcription inhibition involving the regulatory domain of σ subunit, and potentially pinpoint a novel target for development of new antibacterials., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

MucA and MucB are critical negative modulators of sigma factor AlgU and regulate the mucoid conversion of Pseudomonas aeruginosa. Previous studies have revealed that lipid signals antagonize MucA-MucB binding. Here we report the crystal structure of MucB in complex with the periplasmic domain of MucA and polyethylene glycol (PEG), which unveiled an intermediate state preceding the MucA-MucB dissociation. Based on the biochemical experiments, the aliphatic side chain with a polar group was found to be of primary importance for inducing MucA cleavage. These results provide evidence that the hydrophobic cavity of MucB is a primary site for sensing lipid molecules and illustrates the detailed control of conformational switching within MucA-MucB in response to lipophilic effectors.

Formation of a bacterial RNA polymerase (RNAP) holoenzyme by a catalytic core RNAP and a sigma (σ) initiation factor is essential for bacterial viability. As the primary binding site for the housekeeping σ factors, the RNAP clamp helix domain represents an attractive target for novel antimicrobial agent discovery. Previously, we designed a pharmacophore model based on the essential amino acids of the clamp helix, such as R278, R281, and I291 ( Escherichia coli numbering), and identified hit compounds with antimicrobial activity that interfered with the core-σ interactions. In this work, we rationally designed and synthesized a class of triaryl derivatives of one hit compound and succeeded in drastically improving the antimicrobial activity against Streptococcus pneumoniae , with the minimum inhibitory concentration reduced from 256 to 1 μg/mL. Additional characterization of antimicrobial activity, inhibition of transcription, in vitro pharmacological properties, and cytotoxicity of the optimized compounds demonstrated their potential for further development.

The bacterial protein WhiD belongs to the Wbl family of iron-sulfur [Fe-S] proteins present only in the actinomycetes. In Streptomyces coelicolor , it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from in vitro and in vivo experiments with WhiD from Streptomyces venezuelae ( Sv WhiD), which differs from S. coelicolor WhiD ( Sc WhiD) only at the C terminus. We observed that, like Sc WhiD and other Wbl proteins, Sv WhiD binds a [4Fe-4S] cluster that is moderately sensitive to O 2 and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that Sv WhiD exists in a monomer-dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins of Mycobacterium tuberculosis are known to interact with its principal sigma factor SigA. Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate that Sv WhiD interacts with domain 4 of the principal sigma factor of Streptomyces , σ HrdB (σ HrdB 4 ). Using MS, we determined the dissociation constant ( K d ) for the Sv WhiD-σ HrdB 4 complex as ∼0.7 μm, consistent with a relatively tight binding interaction. We found that complex formation was cluster dependent and that a reaction with NO, which was complete at 8-10 NO molecules per cluster, resulted in dissociation into the separate proteins. The Sv WhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O 2 and NO when Sv WhiD was bound to σ HrdB 4 , consistent with protection of the cluster in the complex., (© 2020 Stewart et al.)

RNA polymerases initiate transcription at DNA sequences called promoters. In bacteria, the best conserved promoter feature is the AT-rich -10 element; a sequence essential for DNA unwinding. Further elements, and gene regulatory proteins, are needed to recruit RNA polymerase to the -10 sequence. Hence, -10 elements cannot function in isolation. Many horizontally acquired genes also have a high AT-content. Consequently, sequences that resemble the -10 element occur frequently. As a result, foreign genes are predisposed to spurious transcription. However, it is not clear how RNA polymerase initially recognizes such sequences. Here, we identify a non-canonical promoter element that plays a key role. The sequence, itself a short AT-tract, resides 5 base pairs upstream of otherwise cryptic -10 elements. The AT-tract alters DNA conformation and enhances contacts between the DNA backbone and RNA polymerase., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Transcription activation by cyclic AMP (cAMP) receptor protein (CAP) is the classic paradigm of transcription regulation in bacteria. CAP was suggested to activate transcription on class-II promoters via a recruitment and isomerization mechanism. However, whether and how it modifies RNA polymerase (RNAP) to initiate transcription remains unclear. Here, we report cryo-electron microscopy (cryo-EM) structures of an intact Escherichia coli class-II CAP-dependent transcription activation complex (CAP-TAC) with and without de novo RNA transcript. The structures reveal two distinct architectures of TAC and raise the possibility that CAP binding may induce substantial conformational changes in all the subunits of RNAP and transiently widen the main cleft of RNAP to facilitate DNA promoter entering and formation of the initiation open complex. These structural changes vanish during further RNA transcript synthesis. The observations in this study may reveal a possible on-pathway intermediate and suggest a possibility that CAP activates transcription by inducing intermediate state, in addition to the previously proposed stabilization mechanism., Competing Interests: The authors have declared that no competing interests exist.

In Escherichia coli , SigmaS (σ S ) is the master regulator of the general stress response. The cellular levels of σ S are controlled by transcription, translation and protein stability. The turnover of σ S , by the AAA+ protease (ClpXP), is tightly regulated by a dedicated adaptor protein, termed RssB (Regulator of Sigma S protein B)-which is an atypical member of the response regulator (RR) family. Currently however, the molecular mechanism of σ S recognition and delivery by RssB is only poorly understood. Here we describe the crystal structures of both RssB domains (RssB N and RssB C ) and the SAXS analysis of full-length RssB (both free and in complex with σ S ). Together with our biochemical analysis we propose a model for the recognition and delivery of σ S by this essential adaptor protein. Similar to most bacterial RRs, the N-terminal domain of RssB (RssB N ) comprises a typical mixed (βα) 5 -fold. Although phosphorylation of RssB N (at Asp58) is essential for high affinity binding of σ S , much of the direct binding to σ S occurs via the C-terminal effector domain of RssB (RssB C ). In contrast to most RRs the effector domain of RssB forms a β-sandwich fold composed of two sheets surrounded by α-helical protrusions and as such, shares structural homology with serine/threonine phosphatases that exhibit a PPM/PP2C fold. Our biochemical data demonstrate that this domain plays a key role in both substrate interaction and docking to the zinc binding domain (ZBD) of ClpX. We propose that RssB docking to the ZBD of ClpX overlaps with the docking site of another regulator of RssB, the anti-adaptor IraD. Hence, we speculate that docking to ClpX may trigger release of its substrate through activation of a "closed" state (as seen in the RssB-IraD complex), thereby coupling adaptor docking (to ClpX) with substrate release. This competitive docking to RssB would prevent futile interaction of ClpX with the IraD-RssB complex (which lacks a substrate). Finally, substrate recognition by RssB appears to be regulated by a key residue (Arg117) within the α5 helix of the N-terminal domain. Importantly, this residue is not directly involved in σ S interaction, as σ S binding to the R117A mutant can be restored by phosphorylation. Likewise, R117A retains the ability to interact with and activate ClpX for degradation of σ S , both in the presence and absence of acetyl phosphate. Therefore, we propose that this region of RssB (the α5 helix) plays a critical role in driving interaction with σ S at a distal site.

Regulation of gene expression is critical for Mycobacterium tuberculosis to tolerate stressors encountered during infection and for nonpathogenic mycobacteria such as Mycobacterium smegmatis to survive environmental stressors. Unlike better-studied models, mycobacteria express ∼14% of their genes as leaderless transcripts. However, the impacts of leaderless transcript structures on mRNA half-life and translation efficiency in mycobacteria have not been directly tested. For leadered transcripts, the contributions of 5' untranslated regions (UTRs) to mRNA half-life and translation efficiency are similarly unknown. In M. tuberculosis and M. smegmatis , the essential sigma factor, SigA, is encoded by a transcript with a relatively short half-life. We hypothesized that the long 5' UTR of sigA causes this instability. To test this, we constructed fluorescence reporters and measured protein abundance, mRNA abundance, and mRNA half-life and calculated relative transcript production rates. The sigA 5' UTR conferred an increased transcript production rate, shorter mRNA half-life, and decreased apparent translation rate compared to a synthetic 5' UTR commonly used in mycobacterial expression plasmids. Leaderless transcripts appeared to be translated with similar efficiency as those with the sigA 5' UTR but had lower predicted transcript production rates. A global comparison of M. tuberculosis mRNA and protein abundances failed to reveal systematic differences in protein/mRNA ratios for leadered and leaderless transcripts, suggesting that variability in translation efficiency is largely driven by factors other than leader status. Our data are also discussed in light of an alternative model that leads to different conclusions and suggests leaderless transcripts may indeed be translated less efficiently. IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis , is a major public health problem killing 1.5 million people globally each year. During infection, M. tuberculosis must alter its gene expression patterns to adapt to the stress conditions it encounters. Understanding how M. tuberculosis regulates gene expression may provide clues for ways to interfere with the bacterium's survival. Gene expression encompasses transcription, mRNA degradation, and translation. Here, we used Mycobacterium smegmatis as a model organism to study how 5' untranslated regions affect these three facets of gene expression in multiple ways. We furthermore provide insight into the expression of leaderless mRNAs, which lack 5' untranslated regions and are unusually prevalent in mycobacteria., (Copyright © 2020 Nguyen et al.)

Protein lysine acetylation, one of the most abundant post-translational modifications in eukaryotes, occurs in prokaryotes as well. Despite the evidence of lysine acetylation in bacterial RNA polymerases (RNAPs), its function remains unknown. We found that the housekeeping sigma factor (HrdB) was acetylated throughout the growth of an actinobacterium, Streptomyces venezuelae, and the acetylated HrdB was enriched in the RNAP holoenzyme complex. The lysine (K259) located between 1.2 and 2 regions of the sigma factor, was determined to be the acetylated residue of HrdB in vivo by LC-MS/MS analyses. Specifically, the label-free quantitative analysis revealed that the K259 residues of all the HrdB subunits were acetylated in the RNAP holoenzyme. Using mutations that mimic or block acetylation (K259Q and K259R), we found that K259 acetylation enhances the interaction of HrdB with the RNAP core enzyme as well as the binding activity of the RNAP holoenzyme to target promoters in vivo. Taken together, these findings provide a novel insight into an additional layer of modulation of bacterial RNAP activity., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

All organisms-bacteria, archaea, and eukaryotes-have a transcription initiation factor that contains a structural module that binds within the RNA polymerase (RNAP) active-center cleft and interacts with template-strand single-stranded DNA (ssDNA) in the immediate vicinity of the RNAP active center. This transcription initiation-factor structural module preorganizes template-strand ssDNA to engage the RNAP active center, thereby facilitating binding of initiating nucleotides and enabling transcription initiation from initiating mononucleotides. However, this transcription initiation-factor structural module occupies the path of nascent RNA and thus presumably must be displaced before or during initial transcription. Here, we report four sets of crystal structures of bacterial initially transcribing complexes that demonstrate and define details of stepwise, RNA-extension-driven displacement of the "σ-finger" of the bacterial transcription initiation factor σ. The structures reveal that-for both the primary σ-factor and extracytoplasmic (ECF) σ-factors, and for both 5'-triphosphate RNA and 5'-hydroxy RNA-the "σ-finger" is displaced in stepwise fashion, progressively folding back upon itself, driven by collision with the RNA 5'-end, upon extension of nascent RNA from ∼5 nt to ∼10 nt., Competing Interests: The authors declare no competing interest.

Mycobacterium tuberculosis is equipped with diversified ECF sigma factors that are generally expressed under adverse environmental conditions. Mtb-SigI belongs to the ECF41 family of sigma factor, and no information is available about their expression during stringent response. This study provides the structural insight of Mtb-SigI and the characterization of its C-terminal polypeptide extension. C-terminal site of Mtb-SigI is truncated in two ways: (a) conserved region of C-terminal extension is preserved while the rest of the portion is deleted and (b) complete deletion of C-terminal extension. Each of the wild-type and truncated Mtb-SigI is docked with a β subunit of core RNA polymerase and simulated for 100 ns. Relative binding strength calculated from trajectory analysis reflects that the complete deletion of the C-terminal extension of Mtb-SigI favors interaction with core RNA polymerase. It can be implicated that the C-terminal domain in the wild-type docked complex help flipping of domain 4 of Mtb-SigI and thereby impaired holoenzyme formation. When the C-terminal extension is partially deleted, such flipping of domain 4 of Mtb-SigI diminishes and complete deletion of C-terminal extension promotes holoenzyme formation. In the absence of any sigma factor antagonist, the C-terminal extension of Mtb-SigI might behave as a complex player in transcription regulation. Graphical abstract Role of Mtb-SigI in transcription regulation.

Reiterative transcription is a non-canonical form of RNA synthesis by RNA polymerase in which a ribonucleotide specified by a single base in the DNA template is repetitively added to the nascent RNA transcript. We previously determined the X-ray crystal structure of the bacterial RNA polymerase engaged in reiterative transcription from the pyrG promoter, which contains eight poly-G RNA bases synthesized using three C bases in the DNA as a template and extends RNA without displacement of the promoter recognition σ factor from the core enzyme. In this study, we determined a series of transcript initiation complex structures from the pyrG promoter using soak-trigger-freeze X-ray crystallography. We also performed biochemical assays to monitor template DNA translocation during RNA synthesis from the pyrG promoter and in vitro transcription assays to determine the length of poly-G RNA from the pyrG promoter variants. Our study revealed how RNA slips on template DNA and how RNA polymerase and template DNA determine length of reiterative RNA product. Lastly, we determined a structure of a transcript initiation complex at the pyrBI promoter and proposed an alternative mechanism of RNA slippage and extension requiring the σ dissociation from the core enzyme., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacteriophage T7 infects Escherichia coli and evades the host restriction/modification system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors. Using cryo electron microscopy, we determined the structures of Ocr bound to RNAP. The structures show that an Ocr dimer binds to RNAP in the cleft, where key regions of sigma bind and where DNA resides during transcription synthesis, thus providing a structural basis for the transcription inhibition. Our results reveal the versatility of Ocr in interfering with host systems and suggest possible strategies that could be exploited in adopting DNA mimicry as a basis for forming novel antibiotics., Competing Interests: FY, IK, MJ, XL, DD, MB, XZ No competing interests declared, (© 2020, Ye et al.)

WhiB1 is a monomeric iron-sulfur cluster-containing transcription factor in the WhiB-like family that is widely distributed in actinobacteria including the notoriously persistent pathogen Mycobacterium tuberculosis (M. tuberculosis). WhiB1 plays multiple roles in regulating cell growth and responding to nitric oxide stress in M. tuberculosis, but its underlying mechanism is unclear. Here we report a 1.85 Å-resolution crystal structure of the [4Fe-4S] cluster-bound (holo-) WhiB1 in complex with the C-terminal domain of the σ70-family primary sigma factor σA of M. tuberculosis containing the conserved region 4 (σA4). Region 4 of the σ70-family primary sigma factors is commonly used by transcription factors for gene activation, and holo-WhiB1 has been proposed to activate gene expression via binding to σA4. The complex structure, however, unexpectedly reveals that the interaction between WhiB1 and σA4 is dominated by hydrophobic residues in the [4Fe-4S] cluster binding pocket, distinct from previously characterized canonical σ704-bound transcription activators. Furthermore, we show that holo-WhiB1 represses transcription by interaction with σA4in vitro and that WhiB1 must interact with σA4 to perform its essential role in supporting cell growth in vivo. Together, these results demonstrate that holo-WhiB1 regulates gene expression by a non-canonical mechanism relative to well-characterized σA4-dependent transcription activators., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the essential components dissociate. Our single-molecule fluorescence study unveils that RNA transcript release precedes RNA polymerase (RNAP) dissociation from the DNA template much more often than their concurrent dissociations in intrinsic termination of bacterial transcription. As termination is defined by the release of product RNA from the transcription complex, the subsequent retention of RNAP on DNA constitutes a previously unidentified stage, termed here as recycling. During the recycling stage, post-terminational RNAPs one-dimensionally diffuse on DNA in downward and upward directions, and can initiate transcription again at the original and nearby promoters in the case of retaining a sigma factor. The efficiency of this event, termed here as reinitiation, increases with supplement of a sigma factor. In summary, after releasing RNA product at intrinsic termination, recycling RNAP diffuses on the DNA template for reinitiation most of the time.

Extra Cytoplasmic Function (ECF) σ factor/regulatory protein (anti-σ factor) pairs govern environment mediated changes in gene expression in bacteria. The release of the ECF σ factor from an inactive σ/anti-σ factor complex is triggered by specific environmental stimuli. The free σ factor then associates with the RNA polymerase and drives the expression of genes in its target regulon. Multiple ECF σ/anti-σ pairs ensure calibrated changes in the expression profile by correlating diverse environmental stimuli with changes in the intracellular levels of different ECF σ factors. Specificity in σ/anti-σ factor interaction is thus essential for accurate signal transduction. Here we describe experiments to evaluate interactions between different M. tuberculosis σ and anti-σ proteins in vitro. The interaction parameters suggest that cross-talk between non-cognate σ/anti-σ pairs is likely. The sequence and conformational determinants that govern interaction specificity in a σ/anti-σ complex are not immediately evident due to substantial structural conservation. Sequence-structure analysis of all σ/anti-σ pairs suggest that conserved residues are not the primary determinants of σ/anti-σ interactions-a finding that suggests a potential route to set tolerance limits in interaction specificity. Non-specific σ/anti-σ interactions are likely to be biologically significant as it can contribute to heterogeneity in cellular responses in a bacterial population under less stringent requirements. This finding is relevant for synthetic biology approaches to engineer bacteria using σ/anti-σ transcription initiation modules for diverse applications in biotechnology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In class II transcription activation, the transcription factor normally binds to the promoter near the -35 position and contacts the domain 4 of σ factors (σ 4 ) to activate transcription. However, σ 4 of σ 70 appears to be poorly folded on its own. Here, by fusing σ 4 with the RNA polymerase β-flap-tip-helix, we constructed two σ 4 chimera proteins, one from σ 70 σ 4 70 c and another from σ S σ 4 S c of Klebsiella pneumoniae. The two chimera proteins well folded into a monomeric form with strong binding affinities for -35 element DNA. Determining the crystal structure of σ 4 S c in complex with -35 element DNA revealed that σ 4 S c adopts a similar structure as σ 4 in the Escherichia coli RNA polymerase σ S holoenzyme and recognizes -35 element DNA specifically by several conserved residues from the helix-turn-helix motif. By using nuclear magnetic resonance (NMR), σ 4 70 c was demonstrated to recognize -35 element DNA similar to σ 4 S c . Carr-Purcell-Meiboom-Gill relaxation dispersion analyses showed that the N-terminal helix and the β-flap-tip-helix of σ 4 70 c have a concurrent transient α-helical structure and DNA binding reduced the slow dynamics on σ 4 70 c . Finally, only σ 4 70 c was shown to interact with the response regulator PmrA and its promoter DNA. The chimera proteins are capable of -35 element DNA recognition and can be used for study with transcription factors or other factors that interact with domain 4 of σ factors., (© 2019 Wiley Periodicals, Inc.)

σ S is a master transcription initiation factor that protects bacterial cells from various harmful environmental stresses including antibiotic pressure. Although its mechanism remains unclear, it is known that full activation of σ S -mediated transcription requires a σ S -specific activator, Crl. In this study, we determined a 3.80 Å cryo-EM structure of an Escherichia coli transcription activation complex ( E. coli Crl-TAC) comprising E. coli σ S -RNA polymerase (σ S -RNAP) holoenzyme, Crl, and a nucleic-acid scaffold. The structure reveals that Crl interacts with domain 2 of σ S (σ S 2 ) and the RNAP core enzyme, but does not contact promoter DNA. Results from subsequent hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicate that Crl stabilizes key structural motifs within σ S 2 to promote the assembly of the σ S -RNAP holoenzyme and also to facilitate formation of an RNA polymerase-promoter DNA open complex (RPo). Our study demonstrates a unique DNA contact-independent mechanism of transcription activation, thereby defining a previously unrecognized mode of transcription activation in cells., Competing Interests: JX, KC, LS, JS, LL, LY, CF, GZ, YF, BY, YZ No competing interests declared, (© 2019, Xu et al.)

Alginate production in Pseudomonas aeruginosa is regulated by the alternate σ factor AlgU, which in turn is regulated by the MucABCD system. The anti-σ factor MucA binds AlgU in the cytoplasm and prevents AlgU from binding to the RNA polymerase for transcription. MucB binds MucA in the periplasm and inhibits proteolysis of MucA and subsequent release of AlgU. In this work, we report crystal structures of MucA in complex with AlgU and MucB. A structure of MucB alone reveals the structural changes required for MucA recognition. A unique disulfide bond is identified in MucB, and mutation of this disulfide bond results in a shift from monomer to MucB dimers or tetramers. As MucB tetramers have previously been shown to be unable to bind MucA, this suggests a redox-sensitive stress response mechanism in MucB. The AlgU-MucA structure reveals a conserved σ factor/anti-σ factor complex, but AlgU lacks a disulfide bond conserved in many other σ factors. Our structures reveal the molecular basis for MucA recognition by MucB in the periplasm and AlgU in the cytoplasm, thus providing an important step in understanding the mechanisms that regulate a key signal transduction pathway involved in P. aeruginosa pathogenesis. DATABASE: The atomic coordinates and structure factors for MucA cyto -AlgU, MucB, and MucA peri -MucB have been deposited in the Protein Data Bank (PDB) with the accession code 6IN7, 6IN8, and 6IN9, respectively., (© 2019 Federation of European Biochemical Societies.)

Prokaryotic transcription is one of the most studied biological systems, with relevance to many fields including the development and use of antibiotics, the construction of synthetic gene networks, and the development of many cutting-edge methodologies. Here, we discuss recent structural, biochemical, and single-molecule biophysical studies targeting the mechanisms of transcription initiation in bacteria, including the formation of the open complex, the reaction of initial transcription, and the promoter escape step that leads to elongation. We specifically focus on the mechanisms employed by the RNA polymerase holoenzyme with the housekeeping sigma factor σ 70 . The recent progress provides answers to long-held questions, identifies intriguing new behaviours, and opens up fresh questions for the field of transcription., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In bacteria, a primary σ-factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ-factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative σ-factors are negatively regulated by anti-σ-factors. In Escherichia coli , Salmonella enterica , and many other γ-proteobacteria, the transcription factor Crl positively regulates the alternative σ S -regulon by promoting the association of σ S with RNAP without interacting with promoter DNA. The molecular mechanism for Crl activity is unknown. Here, we determined a single-particle cryo-electron microscopy structure of Crl-σ S -RNAP in an open promoter complex with a σ S -regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of σ S (σ S 2 ), the structure, along with p -benzoylphenylalanine cross-linking, reveals that Crl interacts with a structural element of the RNAP β'-subunit that we call the β'-clamp-toe (β'CT). Deletion of the β'CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-β'CT interaction. We conclude that Crl activates σ S -dependent transcription in part through stabilizing σ S -RNAP by tethering σ S 2 and the β'CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators., Competing Interests: The authors declare no conflict of interest.

The sigma-E transcription factor (σ E TF) can be found in most of the bacteria cells including Bacillus thuringiensis. However, the cellular regulatory mechanisms of these transcription factors in the mass production of δ-endotoxins during sporulation stage are yet to be revealed. In addition, the recognition of DNA towards σ E TF DNA binding motifs that led to the transcription activities is also being poorly studied. Therefore, this work studied the possible DNA binding motifs of σ E TF by utilising in silico approaches. The structure of σ E TF was first built via three different computational methods. A cognate DNA sequence was then docked to the predicted σ E TF DNA-binding motifs. The binding free energy calculated using molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) for triplicate 50 ns simulation of σ E TF-DNA complex revealed favourable binding energy of DNA to σ E TF (average ∆G bind  = -34.57 kcal/mol) mainly driven by non-polar interactions. This study revealed that σ E TF LYS131, ARG133, PHE138, TRP146, ARG222, LYS225 and ARG226 are most likely the key residues upon the binding and recognition of DNA prior to transcription actives. Since determination of genome-regulating protein which recognises specific DNA sequence is important to discriminate between the proteins preferences for different genes, this study might provide some understanding on the possible σ E TF-DNA recognition prior to transcription initiated for the δ-endotoxins production.

Sigma factors are key proteins that mediate the recruitment of RNA polymerase to the promoter regions of genes, for the initiation of bacterial transcription. Multiple sigma factors in a bacterium selectively recognize their cognate promoter sequences, thereby inducing the expression of their own regulons. In this paper, we report the crystal structure of the σ4 domain of Bacillus subtilis SigW bound to the -35 promoter element. Purine-specific hydrogen bonds of the -35 promoter element with the recognition helix α9 of the σ4 domain occurs at three nucleotides of the consensus sequence (G-35, A-34, and G'-31 in G-35A-34A-33A-32C-31C-30T-29). The hydrogen bonds of the backbone with the α7 and α8 of the σ4 domain occurs at G'-30. These results elucidate the structural basis of the selective recognition of the promoter by SigW. In addition, comparison of SigW structures complexed with the -35 promoter element or with anti-sigma RsiW reveals that DNA recognition and anti-sigma factor binding of SigW are mutually exclusive., Competing Interests: The authors have declared that no competing interests exist.

The genomic era along with major advances in high-throughput sequencing technology has led to a rapid expansion of the genomic and consequently the protein sequence space. Bacterial extracytoplasmic function sigma factors have emerged as an important group of signaling proteins in bacteria involved in many regulatory decisions, most notably the adaptation to cell envelope stress. Their wide prevalence and amplification among bacterial genomes has led to sub-group classification and the realization of diverse signaling mechanisms. Mathematical frameworks have been developed to utilize extensive protein sequence alignments to extract co-evolutionary signals of interaction. This has proven useful in a number of different biological fields, including de novo structure prediction, protein-protein partner identification and the elucidation of alternative protein conformations for signal proteins, to name a few. The mathematical tools, commonly referred to under the name 'Direct Coupling Analysis' have now been applied to deduce molecular mechanisms of activation for sub-groups of extracytoplasmic sigma factors adding to previous successes on bacterial two-component signaling proteins. The amplification of signal transduction protein genes in bacterial genomes made them the first to be amenable to this approach but the sequences are available now to aid the molecular microbiologist, no matter their protein pathway of interest., (© 2019 John Wiley & Sons Ltd.)

Bacteria contain sigma (σ) factors that control gene expression in response to various environmental stimuli. The alternative sigma factors σ FpvI and σ PvdS bind specifically to the antisigma factor FpvR. These proteins are an essential component of the pyoverdine-based system for iron uptake in Pseudomonas aeruginosa. Due to the uniqueness of this system, where the activities of both the σ FpvI and σ PvdS sigma factors are regulated by the same antisigma factor, the interactions between the antisigma protein FpvR 20 and the σ FpvI and σ PvdS proteins have been widely studied in vivo. However, difficulties in obtaining soluble, recombinant preparations of the σ FpvI and σ PvdS proteins have limited their biochemical and structural characterizations. In this study, we describe a purification protocol that resulted in the production of soluble, recombinant His 6 -σ FpvI /FpvR 1-67 , His 6 -σ FpvI /FpvR 1-89 , His 6 -σ PvdS /FpvR 1-67 and His 6 -σ PvdS /FpvR 1-89 protein complexes (where FpvR 1-67 and FpvR 1-89 are truncated versions of FpvR 20 ) at high purities and concentrations, appropriate for biophysical analyses by circular dichroism spectroscopy and analytical ultracentrifugation. These results showed the proteins to be folded in solution and led to the determination of the affinities of the protein-protein interactions within the His 6 -σ FpvI /FpvR 1-67 and His 6 -σ PvdS /FpvR 1-67 complexes. A comparison of these values with those previously reported for the His 6 -σ FpvI /FpvR 1-89 and His 6 -σ PvdS /FpvR 1-89 complexes is made., (Copyright © 2019 Elsevier Inc. All rights reserved.)