Oligomerization states of the Mycobacterium tuberculosis RNA polymerase core and holoenzymes.

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 (α2ßß'ω) can also exist as dimers in vitro. Using similar methods, we also show that the holoenzyme (core + σA) 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 σA 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 σA factor.

General transcription factor from Escherichia coli with a distinct mechanism of action.

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) σ70 holoenzyme. Structural and biochemical analysis of CedA bound to RNAP reveal that it bridges distant domains of ß and σ70 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.

Structure of phosphorylated-like RssB, the adaptor delivering σs to the ClpXP proteolytic machinery, reveals an interface switch for activation.

In enterobacteria such as Escherichia coli, the general stress response is mediated by σs, the stationary phase dissociable promoter specificity subunit of RNA polymerase. σs 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-ß5-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 σs-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 σs 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.

Crystal structures of Streptomyces tsukubaensis sigma factor SigG1 and anti-sigma RsfG.

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, σ2 and σ4, 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 σ2, σ4 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 ß-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.

Essential autoproteolysis of bacterial anti-σ factor RsgI for transmembrane signal transduction.

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 ß1 and ß2 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.

Characterization of a transitionally occupied state and thermal unfolding of domain 1.1 of σ A factor of RNA polymerase from Bacillus subtilis.

σ factors are essential parts of bacterial RNA polymerase (RNAP) as they allow to recognize promotor sequences and initiate transcription. Domain 1.1 of vegetative σ factors occupies the primary channel of RNAP and also prevents binding of the σ factor to promoter DNA alone. Here, we show that domain 1.1 of Bacillus subtilis σ A exists in more structurally distinct variants in dynamic equilibrium. The major conformation at room temperature is represented by a previously reported well-folded structure solved by nuclear magnetic resonance (NMR), but 4% of the protein molecules are present in a less thermodynamically favorable state. We show that this population increases with temperature and we predict its significant elevation at higher but still biologically relevant temperatures. We characterized the minor state of the domain 1.1 using specialized methods of NMR. We found that, in contrast to the major state, the detected minor state is partially unfolded. Its propensity to form secondary structure elements is especially decreased for the first and third α helices, while the second α helix and ß strand close to the C-terminus are more stable. We also analyzed thermal unfolding of the domain 1.1 and performed functional experiments with full length σ A and its shortened version lacking domain 1.1 ( σ A _ Δ 1.1 ). The results revealed that while full length σ A increases transcription activity of RNAP with increasing temperature, transcription with σ A _ Δ 1.1 remains constant. In summary, this study reveals conformational dynamics of domain 1.1 and provides a basis for studies of its interaction with RNAP and effects on transcription regulation.

Structural basis of DNA binding by the WhiB-like transcription factor WhiB3 in Mycobacterium tuberculosis.

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 (σA4) 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 σA4 to bind DNA and regulate transcription is unclear. Here we determined crystal structures of the WhiB3:σA4 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:σA4 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 σA4 and engaging with DNA via the subclass-specific structural motif, distinct from the modes of DNA interaction by WhiB1 and WhiB7.

An SI3-σ arch stabilizes cyanobacteria transcription initiation complex.

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 σA (σ2) 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.

A general mechanism for transcription bubble nucleation in bacteria.

Bacterial transcription initiation requires σ factors for nucleation of the transcription bubble. The canonical housekeeping σ factor, σ70, nucleates DNA melting via recognition of conserved bases of the promoter -10 motif, which are unstacked and captured in pockets of σ70. By contrast, the mechanism of transcription bubble nucleation and formation during the unrelated σN-mediated transcription initiation is poorly understood. Herein, we combine structural and biochemical approaches to establish that σN, like σ70, 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.

Clamp Interactions with +3/+6 Duplex and Upstream-to-Downstream Allosteric Effects in Late Steps of Forming a Stable RNA Polymerase-Promoter Open Complex.

Stable 37 °C open complexes (OC) of E. coli RNA polymerase (RNAP) at λPR 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 (I2) intermediates for promoters with different combinations of λPR (L) and T7A1 (T) discriminators, core promoters and UP elements. I2 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 ΔG3o for I2 â†’ 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 ΔG3o values similar (approximately -4 kcal). Urea reduces OC lifetime greatly by affecting ΔG3o. We deduce that urea acts by disfavoring coupled folding of key elements of the ß'-clamp, that I2 is an open-clamp OC, and that clamp-closing in I2 â†’ stable OC involves coupled folding. Differences in ΔG3o between downstream-truncated and full-length promoters yield contributions to ΔG3o from interactions with downstream mobile elements (DME) including ß-lobe and ß'-jaw, more favorable for L-discriminator than for T-discriminator promoters. We deduce how competition between far-downstream DNA and σ70 region 1.1 affects ΔG3o values. We discuss variant-specific ΔG3o 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 I2 â†’ stable OC.

Identification and Characterization of the Alternative σ28 Factor in Treponema denticola.

FliA (also known as σ28), a member of the bacterial σ70 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 σ28 of Escherichia coli, binds to σ28-dependent promoters, and can functionally replace the σ28 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 σ4 region of σ28, are indispensable for the binding activity of TDE2683 to the σ28-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 σ28 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 σ28 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 σ28 in spirochetes.

Deciphering the role of the two conserved motifs of the ECF41 family σ factor in the autoregulation of its own promoter in Azospirillum brasilense Sp245.

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 σ2 and σ4 , 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 σ2 -σ4 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.

The structure of the Clostridium thermocellum RsgI9 ectodomain provides insight into the mechanism of biomass sensing.

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.

Structural analysis of alternate sigma factor ComX with RpoC, RpoB and its cognate CIN promoter reveals a distinctive promoter melting mechanism.

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 ß subunit and RpoC ß' 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.

Structural basis of transcription activation by the global regulator Spx.

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.

Phospho-dependent signaling during the general stress response by the atypical response regulator and ClpXP adaptor RssB.

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 BeF3- . 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.

Transcription activation by a sliding clamp.

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.

Alternative Sigma Factor of Staphylococcus aureus Interacts with the Cognate Antisigma Factor Primarily Using Its Domain 3.

σB, 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. σB, a σ70-like sigma factor, harbors three conserved domains designated σB2, σB3, and σB4. To better understand the interaction between RsbW and σB or its domains, we have studied their recombinant forms, rRsbW, rσB, rσB2, rσB3, and rσB4, using different probes. The results show that none of the rσB domains, unlike rσB, showed binding to a cognate DNA in the presence of a core RNA polymerase. However, both rσB2 and rσB3, like rσB, interacted with rRsbW, and the order of their rRsbW binding affinity looks like rσB > rσB3 > rσB2. Furthermore, the reaction between rRsbW and rσB or rσB3 was exothermic and occurred spontaneously. rRsbW and rσB3 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-σB3 complex that has supported our experimental results indicated the binding of rσB3 at the putative dimeric interface of RsbW. A genetic study shows that the tentative dimer-forming region of RsbW is crucial for preserving its rσB binding ability, serine kinase activity, and dimerization ability. Additionally, a urea-induced equilibrium unfolding study indicated a notable thermodynamic stabilization of σB3 in the presence of RsbW. Possible implications of the stabilization data in drug discovery were discussed at length.

Structural basis of ribosomal RNA transcription regulation.

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) σ70 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 ß' 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 σ1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding also opens the DNA loading gate, which is not coupled to σ1.1 ejection and impedes open complex formation. These results provide a molecular basis for the exceptionally active rRNA transcription and its vulnerability to DksA/ppGpp.

RssB-mediated σS Activation is Regulated by a Two-Tier Mechanism via Phosphorylation and Adaptor Protein - IraD.

Regulation of bacterial stress responding σS is a sophisticated process and mediated by multiple interacting partners. Controlled proteolysis of σS is regulated by RssB which maintains minimal level of σS during exponential growth but then elevates σS 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-σS interaction remains elusive. Here, we demonstrated the impact of phosphorylation on the activity of RssB and built the RssB-σS complex model. Importantly, we showed that the phosphorylation site - D58 is at the interface of RssB-σS complex. Hence, mutation or phosphorylation of D58 would weaken the interaction of RssB with σS. We found that the anti-adaptor protein IraD has higher affinity than σS to RssB and its binding interface on RssB overlaps with that for σS. And IraD-RssB complex is preferred over RssB-σS in solution, regardless of the phosphorylation state of RssB. Our study suggests that RssB possesses a two-tier mechanism for regulating σS. 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.