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.

Mechanisms of DNA opening revealed in AAA+ transcription complex structures.

Gene transcription is carried out by RNA polymerase (RNAP) and requires the conversion of the initial closed promoter complex, where DNA is double stranded, to a transcription-competent open promoter complex, where DNA is opened up. In bacteria, RNAP relies on σ factors for its promoter specificities. Using a special form of sigma factor (σ54), which forms a stable closed complex and requires its activator that belongs to the AAA+ ATPases (ATPases associated with diverse cellular activities), we obtained cryo-electron microscopy structures of transcription initiation complexes that reveal a previously unidentified process of DNA melting opening. The σ54 amino terminus threads through the locally opened up DNA and then becomes enclosed by the AAA+ hexameric ring in the activator-bound intermediate complex. Our structures suggest how ATP hydrolysis by the AAA+ activator could remove the σ54 inhibition while helping to open up DNA, using σ54 amino-terminal peptide as a pry bar.

Bacterial Enhancer Binding Proteins-AAA+ Proteins in Transcription Activation.

Bacterial enhancer-binding proteins (bEBPs) are specialised transcriptional activators. bEBPs are hexameric AAA+ ATPases and use ATPase activities to remodel RNA polymerase (RNAP) complexes that contain the major variant sigma factor, σ54 to convert the initial closed complex to the transcription competent open complex. Earlier crystal structures of AAA+ domains alone have led to proposals of how nucleotide-bound states are sensed and propagated to substrate interactions. Recently, the structure of the AAA+ domain of a bEBP bound to RNAP-σ54-promoter DNA was revealed. Together with structures of the closed complex, an intermediate state where DNA is partially loaded into the RNAP cleft and the open promoter complex, a mechanistic understanding of how bEBPs use ATP to activate transcription can now be proposed. This review summarises current structural models and the emerging understanding of how this special class of AAA+ proteins utilises ATPase activities to allow σ54-dependent transcription initiation.

Mechanisms of σ54-Dependent Transcription Initiation and Regulation.

Cellular RNA polymerase is a multi-subunit macromolecular assembly responsible for gene transcription, a highly regulated process conserved from bacteria to humans. In bacteria, sigma factors are employed to mediate gene-specific expression in response to a variety of environmental conditions. The major variant σ factor, σ54, has a specific role in stress responses. Unlike σ70-dependent transcription, which often can spontaneously proceed to initiation, σ54-dependent transcription requires an additional ATPase protein for activation. As a result, structures of a number of distinct functional states during the dynamic process of transcription initiation have been captured using the σ54 system with both x-ray crystallography and cryo electron microscopy, furthering our understanding of σ54-dependent transcription initiation and DNA opening. Comparisons with σ70 and eukaryotic polymerases reveal unique and common features during transcription initiation.

Role of the σ54 Activator Interacting Domain in Bacterial Transcription Initiation.

Bacterial sigma factors are subunits of RNA polymerase that direct the holoenzyme to specific sets of promoters in the genome and are a central element of regulating transcription. Most polymerase holoenzymes open the promoter and initiate transcription rapidly after binding. However, polymerase containing the members of the σ54 family must be acted on by a transcriptional activator before DNA opening and initiation occur. A key domain in these transcriptional activators forms a hexameric AAA+ ATPase that acts through conformational changes brought on by ATP hydrolysis. Contacts between the transcriptional activator and σ54 are primarily made through an N-terminal σ54 activator interacting domain (AID). To better understand this mechanism of bacterial transcription initiation, we characterized the σ54 AID by NMR spectroscopy and other biophysical methods and show that it is an intrinsically disordered domain in σ54 alone. We identified a minimal construct of the Aquifex aeolicus σ54 AID that consists of two predicted helices and retains native-like binding affinity for the transcriptional activator NtrC1. Using the NtrC1 ATPase domain, bound with the non-hydrolyzable ATP analog ADP-beryllium fluoride, we studied the NtrC1-σ54 AID complex using NMR spectroscopy. We show that the σ54 AID becomes structured after associating with the core loops of the transcriptional activators in their ATP state and that the primary site of the interaction is the first predicted helix. Understanding this complex, formed as the first step toward initiation, will help unravel the mechanism of σ54 bacterial transcription initiation.

Transcription Regulation and Membrane Stress Management in Enterobacterial Pathogens.

Transcription regulation in a temporal and conditional manner underpins the lifecycle of enterobacterial pathogens. Upon exposure to a wide array of environmental cues, these pathogens modulate their gene expression via the RNA polymerase and associated sigma factors. Different sigma factors, either involved in general 'house-keeping' or specific responses, guide the RNA polymerase to their cognate promoter DNAs. The major alternative sigma54 factor when activated helps pathogens manage stresses and proliferate in their ecological niches. In this chapter, we review the function and regulation of the sigma54-dependent Phage shock protein (Psp) system-a major stress response when Gram-negative pathogens encounter damages to their inner membranes. We discuss the recent development on mechanisms of gene regulation, signal transduction and stress mitigation in light of different biophysical and biochemical approaches.

Mutations in RNA Polymerase Bridge Helix and Switch Regions Affect Active-Site Networks and Transcript-Assisted Hydrolysis.

In bacterial RNA polymerase (RNAP), the bridge helix and switch regions form an intricate network with the catalytic active centre and the main channel. These interactions are important for catalysis, hydrolysis and clamp domain movement. By targeting conserved residues in Escherichia coli RNAP, we are able to show that functions of these regions are differentially required during σ(70)-dependent and the contrasting σ(54)-dependent transcription activations and thus potentially underlie the key mechanistic differences between the two transcription paradigms. We further demonstrate that the transcription factor DksA directly regulates σ(54)-dependent activation both positively and negatively. This finding is consistent with the observed impacts of DksA on σ(70)-dependent promoters. DksA does not seem to significantly affect RNAP binding to a pre-melted promoter DNA but affects extensively activity at the stage of initial RNA synthesis on σ(54)-regulated promoters. Strikingly, removal of the σ(54) Region I is sufficient to invert the action of DksA (from stimulation to inhibition or vice versa) at two test promoters. The RNAP mutants we generated also show a strong propensity to backtrack. These mutants increase the rate of transcript-hydrolysis cleavage to a level comparable to that seen in the Thermus aquaticus RNAP even in the absence of a non-complementary nucleotide. These novel phenotypes imply an important function of the bridge helix and switch regions as an anti-backtracking ratchet and an RNA hydrolysis regulator.

TRANSCRIPTION. Structures of the RNA polymerase-σ54 reveal new and conserved regulatory strategies.

Transcription by RNA polymerase (RNAP) in bacteria requires specific promoter recognition by σ factors. The major variant σ factor (σ(54)) initially forms a transcriptionally silent complex requiring specialized adenosine triphosphate-dependent activators for initiation. Our crystal structure of the 450-kilodalton RNAP-σ(54) holoenzyme at 3.8 angstroms reveals molecular details of σ(54) and its interactions with RNAP. The structure explains how σ(54) targets different regions in RNAP to exert its inhibitory function. Although σ(54) and the major σ factor, σ(70), have similar functional domains and contact similar regions of RNAP, unanticipated differences are observed in their domain arrangement and interactions with RNAP, explaining their distinct properties. Furthermore, we observe evolutionarily conserved regulatory hotspots in RNAPs that can be targeted by a diverse range of mechanisms to fine tune transcription.

Hypothetical protein CT398 (CdsZ) interacts with σ(54) (RpoN)-holoenzyme and the type III secretion export apparatus in Chlamydia trachomatis.

A significant challenge to bacteriology is the relatively large proportion of proteins that lack sufficient sequence similarity to support functional annotation (i.e. hypothetical proteins). The aim of this study was to apply protein structural homology to gain insights into a candidate protein of unknown function (CT398) within the medically important, obligate intracellular bacterium Chlamydia trachomatis. C. trachomatis is a major human pathogen responsible for numerous infections throughout the world that can lead to blindness and infertility. A 2.12 Å crystal structure of hypothetical protein CT398 was determined that was comprised of N-terminal coiled-coil and C-terminal Zn-ribbon domains. The structure of CT398 displayed a high degree of structural similarity to FlgZ (Flagellar-associated zinc-ribbon domain protein) from Helicobacter pylori. This observation directed analyses of candidate protein partners of CT398, revealing interactions with two paralogous type III secretion system (T3SS) ATPase-regulators (CdsL and FliH) and the alternative sigma factor RpoN (σ(54) ). Furthermore, genetic introduction of a conditional expression, affinity-tagged construct into C. trachomatis enabled the purification of a CT398-RpoN-holoenzyme complex, suggesting a potential role for CT398 in modulating transcriptional activity during infection. The interactions reported here, in tandem with previous FlgZ studies in H. pylori, indicate that CT398 functions as a regulator of several key areas of chlamydial biology throughout the developmental cycle. Accordingly, we propose that CT398 be named CdsZ (Contact-dependent secretion-associated zinc-ribbon domain protein).

A perspective on the enhancer dependent bacterial RNA polymerase.

Here we review recent findings and offer a perspective on how the major variant RNA polymerase of bacteria, which contains the sigma54 factor, functions for regulated gene expression. We consider what gaps exist in our understanding of its genetic, biochemical and biophysical functioning and how they might be addressed.

Basal Body Structures Differentially Affect Transcription of RpoN- and FliA-Dependent Flagellar Genes in Helicobacter pylori.

UNLABELLED: Flagellar biogenesis in Helicobacter pylori is regulated by a transcriptional hierarchy governed by three sigma factors, RpoD (σ(80)), RpoN (σ(54)), and FliA (σ(28)), that temporally coordinates gene expression with the assembly of the flagellum. Previous studies showed that loss of flagellar protein export apparatus components inhibits transcription of flagellar genes. The FlgS/FlgR two-component system activates transcription of RpoN-dependent genes though an unknown mechanism. To understand better the extent to which flagellar gene regulation is coupled to flagellar assembly, we disrupted flagellar biogenesis at various points and determined how these mutations affected transcription of RpoN-dependent (flaB and flgE) and FliA-dependent (flaA) genes. The MS ring (encoded by fliF) is one of the earliest flagellar structures assembled. Deletion of fliF resulted in the elimination of RpoN-dependent transcripts and an ∼4-fold decrease in flaA transcript levels. FliH is a cytoplasmic protein that functions with the C ring protein FliN to shuttle substrates to the export apparatus. Deletions of fliH and genes encoding C ring components (fliM and fliY) decreased transcript levels of flaB and flgE but had little or no effect on transcript levels of flaA. Transcript levels of flaB and flgE were elevated in mutants where genes encoding rod proteins (fliE and flgBC) were deleted, while transcript levels of flaA was reduced ∼2-fold in both mutants. We propose that FlgS responds to an assembly checkpoint associated with the export apparatus and that FliH and one or more C ring component assist FlgS in engaging this flagellar structure. IMPORTANCE: The mechanisms used by bacteria to couple transcription of flagellar genes with assembly of the flagellum are poorly understood. The results from this study identified components of the H. pylori flagellar basal body that either positively or negatively affect expression of RpoN-dependent flagellar genes. Some of these basal body proteins may interact directly with regulatory proteins that control transcription of the H. pylori RpoN regulon, a hypothesis that can be tested by examining protein-protein interactions in vitro.

In vitro and in vivo methodologies for studying the Sigma 54-dependent transcription.

Here we describe approaches and methods to assaying in vitro the major variant bacterial sigma factor, Sigma 54 (σ(54)), in a purified system. We include the complete transcription system, binding interactions between σ54 and its activators, as well as the self-assembly and the critical ATPase activity of the cognate activators which serve to remodel the closed promoter complexes. We also present in vivo methodologies that are used to study the impact of physiological processes, metabolic states, global signalling networks, and cellular architecture on the control of σ(54)-dependent gene expression.

DNA recognition by a σ(54) transcriptional activator from Aquifex aeolicus.

Transcription initiation by bacterial σ(54)-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain (DBD). The structurally characterized DBDs from activators all belong to the Fis (factor for inversion stimulation) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DBD of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ(54) activators. Two NtrC4-binding sites were identified upstream (-145 and -85bp) from the start of the lpxC gene, which is responsible for the first committed step in lipid A biosynthesis. This is the first experimental evidence for σ(54) regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the -145-binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homolog, Fis. The greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base-specific contacts contributing to affinity than for Fis.

Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies.

Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ(70) or σ(54), that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ(54) version controls gene expression in response to stress, often mediating pathogenicity. Its activator proteins are members of the AAA+ superfamily and use adenosine triphosphate (ATP) hydrolysis to remodel initially auto-inhibited holoenzyme promoter complexes. We have mapped this remodeling using single-molecule fluorescence spectroscopy. Initial remodeling is nucleotide-independent and driven by binding both ssDNA during promoter melting and activator. However, DNA loading into the RNA polymerase active site depends on co-operative ATP hydrolysis by the activator. Although the coupled promoter recognition and melting steps may be conserved between σ(70) and σ(54), the domain movements of the latter have evolved to require an activator ATPase.

Subunit dynamics and nucleotide-dependent asymmetry of an AAA(+) transcription complex.

Bacterial enhancer binding proteins (bEBPs) are transcription activators that belong to the AAA(+) protein family. They form higher-order self-assemblies to regulate transcription initiation at stress response and pathogenic promoters. The precise mechanism by which these ATPases utilize ATP binding and hydrolysis energy to remodel their substrates remains unclear. Here we employed mass spectrometry of intact complexes to investigate subunit dynamics and nucleotide occupancy of the AAA(+) domain of one well-studied bEBP in complex with its substrate, the σ(54) subunit of RNA polymerase. Our results demonstrate that the free AAA(+) domain undergoes significant changes in oligomeric states and nucleotide occupancy upon σ(54) binding. Such changes likely correlate with one transition state of ATP and are associated with an open spiral ring formation that is vital for asymmetric subunit function and interface communication. We confirmed that the asymmetric subunit functionality persists for open promoter complex formation using single-chain forms of bEBP lacking the full complement of intact ATP hydrolysis sites. Outcomes reconcile low- and high-resolution structures and yield a partial sequential ATP hydrolysis model for bEBPs.

Crystallization and preliminary X-ray analysis of the ATPase domain of the σ(54)-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP-BeFx.

One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ(70) RNA polymerase (RNAP), σ(54) RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP-BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators.

Evidence for self-association of the alternative sigma factor σ54.

Sigma factor σ(54) has a distinct modus operandi for mediating the activation of bacterial RNA polymerase (RNAP) at promoter recognition motifs 12 and 24 bp upstream from transcription start sites. σ(54) was thought to act as monomer in all transcription steps. However, we provide evidence that σ(54) of Pseudomonas putida interacts stably with itself. The interface between monomers involves contacts in σ(54) regions I and III, sequences that play key roles in the transcription activation of σ(54)-RNAP holoenzyme. These roles include interactions with activator proteins and the -12 and -24 motifs. In particular, we detected inter-monomer contacts between region I, and between region I and the C-terminal portion of region III. Our results suggest a new auto-antagonistic regulatory state of σ(54).

Structural mechanism of GAF-regulated σ(54) activators from Aquifex aeolicus.

The σ subunits of bacterial RNA polymerase occur in many variant forms and confer promoter specificity to the holopolymerase. Members of the σ(54) family of σ subunits require the action of a 'transcriptional activator' protein to open the promoter and initiate transcription. The activator proteins undergo regulated assembly from inactive dimers to hexamers that are active ATPases. These contact σ(54) directly and, through ATP hydrolysis, drive a conformational change that enables promoter opening. σ(54) activators use several different kinds of regulatory domains to respond to a wide variety of intracellular signals. One common regulatory module, the GAF domain, is used by σ(54) activators to sense small-molecule ligands. The structural basis for GAF domain regulation in σ(54) activators has not previously been reported. Here, we present crystal structures of GAF regulatory domains for Aquifex aeolicus σ(54) activators NifA-like homolog (Nlh)2 and Nlh1 in three functional states-an 'open', ATPase-inactive state; a 'closed', ATPase-inactive state; and a 'closed', ligand-bound, ATPase-active state. We also present small-angle X-ray scattering data for Nlh2-linked GAF-ATPase domains in the inactive state. These GAF domain dimers regulate σ(54) activator proteins by holding the ATPase domains in an inactive dimer conformation. Ligand binding of Nlh1 dramatically remodels the GAF domain dimer interface, disrupting the contacts with the ATPase domains. This mechanism has strong parallels to the response to phosphorylation in some two-component regulated σ(54) activators. We describe a structural mechanism of GAF-mediated enzyme regulation that appears to be conserved among humans, plants, and bacteria.

A common feature from different subunits of a homomeric AAA+ protein contacts three spatially distinct transcription elements.

Initiation of σ(54)-dependent transcription requires assistance to melt DNA at the promoter site but is impeded by numerous protein-protein and nucleo-protein interactions. To alleviate these inhibitory interactions, hexameric bacterial enhancer binding proteins (bEBP), a subset of the ATPases associated with various cellular activities (AAA+) protein family, are required to remodel the transcription complex using energy derived from ATP hydrolysis. However, neither the process of energy conversion nor the internal architecture of the closed promoter complex is well understood. Escherichia coli Phage shock protein F (PspF), a well-studied bEBP, contains a surface-exposed loop 1 (L1). L1 is key to the energy coupling process by interacting with Region I of σ(54) (σ(54)(RI)) in a nucleotide dependent manner. Our analyses uncover new levels of complexity in the engagement of a multimeric bEBP with a basal transcription complex via several L1s. The mechanistic implications for these multivalent L1 interactions are elaborated in the light of available structures for the bEBP and its target complexes.

Lipase A gene transcription in Pseudomonas alcaligenes is under control of RNA polymerase σ54 and response regulator LipR.

Initial analysis has shown that the transcription of the Pseudomonas alcaligenes lipA gene, which encodes an extracellular lipase, is governed by the LipQR two-component system consisting of sensor kinase LipQ and DNA-binding regulator LipR. This study further analyzes lipA gene expression and demonstrates that the RNA polymerase σ54 is involved in the transcription. Purified LipR has an ATPase activity that is stimulated by the presence of lipA promoter DNA. Surface plasmon resonance measurements with purified and in vitro phosphorylated LipR reveal that phosphorylation of LipR is required for specific binding to the upstream activating sequence of the lipA promoter. Furthermore, mass spectrometric analysis combined with mutagenesis demonstrates that Asp52 is the phosphorylated aspartate. This analysis exposes LipR as a prominent member of the growing family of bacterial enhancer-binding proteins.