Abstract: Genetic information in the DNA is accessed by the molecular machine RNA polymerase following a highly conserved process - invariably involving the transition between double and single stranded DNA states. In the case of the bacterial enhancer dependent RNA polymerase (which is essential for adaptive responses and bacterial pathogenesis), the DNA melting event depends on specialised hexameric AAA+ ATPase activators. Involvement of such factors in transcription was demonstrated 25 years ago, but why these activators need to be hexameric, whether all the subunits operate identically, what is the contribution of each of the six subunits within the hexamer (structural, functional or both) and how many active subunits are required for transcription activation remain open questions. Using engineered single-chain polypeptides covalently linking two or three subunits of the activator (allowing he subunit distribution within a hexamer to be fixed), we now show that (i) individual subunits have differential contributions to the oligomer's activities and (ii) only a fraction of the subunits within the hexameric ATPase is directly required for gene activation. We establish that nucleotide dependent co-ordination across three subunits of the hexameric bacterial Enhancer Binding Proteins (bEBP) is necessary for engagement and remodelling of the closed complex (RPc). Outcomes revealed features of bEBP, distinguishing their mode of action from fully processive AAA+ proteins or from simple bimodal switches. We now propose that the hexamer functions with asymmetric organisation, potentially involving a split planar (open-ring) or spiral character.
Abstract: The bacterial phage shock protein (Psp) response functions to help cells manage the impacts of agents impairing cell membrane function. The system has relevance to biotechnology and to medicine. Originally discovered in Escherichia coli, Psp proteins and homologues are found in Gram-positive and Gram-negative bacteria, in archaea and in plants. Study of the E. coli and Yersinia enterocolitica Psp systems provides insights into how membrane-associated sensory Psp proteins might perceive membrane stress, signal to the transcription apparatus and use an ATP-hydrolysing transcription activator to produce effector proteins to overcome the stress. Progress in understanding the mechanism of signal transduction by the membrane-bound Psp proteins, regulation of the psp gene-specific transcription activator and the cell biology of the system is presented and discussed. Many features of the action of the Psp system appear to be dominated by states of self-association of the master effector, PspA, and the transcription activator, PspF, alongside a signalling pathway that displays strong conditionality in its requirement.
Abstract: Homohexameric ring AAA(+) ATPases are found in all kingdoms of life and are involved in all cellular processes. To accommodate the large spectrum of substrates, the conserved AAA(+) core has become specialized through the insertion of specific substrate-binding motifs. Given their critical roles in cellular function, understanding the nucleotide-driven mechanisms of action is of wide importance. For one type of member AAA(+) protein (phage shock protein F, PspF), we identified and established the functional significance of strategically placed arginine and glutamate residues that form interacting pairs in response to nucleotide binding. We show that these interactions are critical for "cis" and "trans" subunit communication, which support coordination between subunits for nucleotide-dependent substrate remodeling. Using an allele-specific suppression approach for ATPase and substrate remodeling, we demonstrate that the targeted residues directly interact and are unlikely to make any other pairwise critical interactions. We then propose a mechanistic rationale by which the nucleotide-bound state of adjacent subunits can be sensed without direct involvement of R-finger residues. As the structural AAA(+) core is conserved, we propose that the functional networks established here could serve as a template to identify similar residue pairs in other AAA(+) proteins.
Abstract: ATP hydrolysis-dependent molecular machines and motors often drive regulated conformational transformations in cell signaling and gene regulation complexes. Conformational reorganization of a gene regulation complex containing the major variant form of bacterial RNA polymerase (RNAP), Esigma(54), requires engagement with its cognate ATP-hydrolyzing activator protein. Importantly, this activated RNAP is essential for a number of adaptive responses, including those required for bacterial pathogenesis. Here we characterize the initial encounter between the enhancer-dependent Esigma(54) and its cognate activator AAA+ ATPase protein, before ADP+P(i) formation, using a small primed RNA (spRNA) synthesis assay. The results show that in a prehydrolysis state, sufficient activator-dependent rearrangements in Esigma(54) have occurred to allow engagement of the RNAP active site with single-stranded promoter DNA to support spRNA synthesis, but not to melt the promoter DNA. This catalytically competent transcription intermediate has similarity with the open promoter complex, in that the RNAP dynamics required for DNA scrunching should be occurring. Significantly, this work highlights that prehydrolysis states of ATPases are functionally important in the molecular transformations they drive.
Abstract: ATP-driven remodelling of initial RNA polymerase (RNAP) promoter complexes occurs as a major post recruitment strategy used to control gene expression. Using a model-enhancer-dependent bacterial system (sigma54-RNAP, Esigma54) and a slowly hydrolysed ATP analogue (ATPgammaS), we provide evidence for a nucleotide-dependent temporal pathway leading to DNA melting involving a small set of sigma54-DNA conformational states. We demonstrate that the ATP hydrolysis-dependent remodelling of Esigma54 occurs in at least two distinct temporal steps. The first detected remodelling phase results in changes in the interactions between the promoter specificity sigma54 factor and the promoter DNA. The second detected remodelling phase causes changes in the relationship between the promoter DNA and the core RNAP catalytic beta/beta' subunits, correlating with the loading of template DNA into the catalytic cleft of RNAP. It would appear that, for Esigma54 promoters, loading of template DNA within the catalytic cleft of RNAP is dependent on fast ATP hydrolysis steps that trigger changes in the beta' jaw domain, thereby allowing acquisition of the open complex status.
Abstract: Bacterial RNA polymerase (RNAP) containing the major variant sigma(54) factor forms open promoter complexes in a reaction in which specialized activator proteins hydrolyse ATP. Here we probe binding interactions between sigma(54)-RNAP (Esigma(54)) and the ATPases associated with various cellular activities (AAA+) domain of the Escherichia coli activator protein, PspF, using nucleotide-metal fluoride (BeF and AlF) analogues representing ground and transition states of ATP, which allow complexes (that are otherwise too transient with ATP) to be captured. We show that the organization and functionality of the ADP-BeF- and ADP-AlF-dependent complexes greatly overlap. Our data support an activation pathway in which the initial ATP-dependent binding of the activator to the Esigma(54) closed complex results in the re-organization of Esigma(54) with respect to the transcription start-site. However, the nucleotide-dependent binding interactions between the activator and the Esigma(54) closed complex are in themselves insufficient for forming open promoter complexes when linear double-stranded DNA is present in the initial closed complex.
Abstract: sigma(54)-dependent transcription requires activation by bacterial enhancer binding proteins (bEBPs). bEBPs are members of the AAA+ (ATPases associated with various cellular activities) protein family and typically form hexameric structures that are crucial for their ATPase activity. The precise mechanism by which the energy derived from ATP hydrolysis is coupled to biological output has several unknowns. Here we use Escherichia coli PspF, a model bEBP involved in the transcription of stress response genes (psp operon), to study determinants of its contact features with the closed promoter complex. We demonstrate that substitution of a highly conserved phenylalanine (F85) residue within the L1 loop GAFTGA motif affects (i) the ATP hydrolysis rate of PspF, demonstrating the link between L1 and the nucleotide binding pocket; (ii) the internal organization of the hexameric ring; and (iii) sigma(54) interactions. Importantly, we provide evidence for a close relationship between F85 and the -12 DNA fork junction structure, which may contribute to key interactions during the energy coupling step and the subsequent remodelling of the Esigma(54) closed complex. The functionality of F85 is distinct from that of other GAFTGA residues, especially T86 where in contrast to F85 a clean uncoupling phenotype is observed.
Abstract: To survive and colonise their various environments, including those used during infection, bacteria have developed a variety of adaptive systems. Amongst these is phage shock protein (Psp) response, which can be induced in Escherichia coli upon filamentous phage infection (specifically phage secretin pIV) and by other membrane-damaging agents. The E. coli Psp system comprises seven proteins, of which PspA is the central component. PspA is a bifunctional protein that is directly involved in (i) the negative regulation of the psp-specific transcriptional activator PspF and (ii) the maintenance of membrane integrity in a mechanism proposed to involve the formation of a 36-mer ring complex. Here we established that the PspA negative regulation of PspF ATPase activity is the result of a cooperative inhibition. We present biochemical evidence showing that an inhibitory PspA-PspF regulatory complex, which has significantly reduced PspF ATPase activity, is composed of around six PspF subunits and six PspA subunits, suggesting that PspA exists in at least two different oligomeric assemblies. We now establish that all four putative helical domains of PspA are critical for the formation of the 36-mer. In contrast, not all four helical domains are required for the formation of the inhibitory PspA-PspF complex. Since a range of initial PspF oligomeric states permit formation of the apparent PspA-PspF dodecameric assembly, we conclude that PspA and PspF demonstrate a strong propensity to self-assemble into a single defined heteromeric regulatory complex.
Abstract: bEBPs (bacterial enhancer-binding proteins) are AAA+ (ATPase associated with various cellular activities) transcription activators that activate gene transcription through a specific bacterial sigma factor, sigma(54). Sigma(54)-RNAP (RNA polymerase) binds to promoter DNA sites and forms a stable closed complex, unable to proceed to transcription. The closed complex must be remodelled using energy from ATP hydrolysis provided by bEBPs to melt DNA and initiate transcription. Recently, large amounts of structural and biochemical data have produced insights into how ATP hydrolysis within the active site of bEBPs is coupled to the re-modelling of the closed complex. In the present article, we review some of the key nucleotides, mutations and techniques used and how they have contributed towards our understanding of the function of bEBPs.
Abstract: Mechanochemical proteins rely on ATP hydrolysis to establish the different functional states required for their biological output. Studying the transient functional intermediate states these proteins adopt as they progress through the ATP hydrolysis cycle is key to understanding the molecular basis of their mechanism. Many of these intermediates have been successfully 'trapped' and functionally characterised using ATP analogues. Here, we present a new nucleotide analogue, AMP-AlF(x), which traps PspF, a bacterial enhancer binding protein, in a stable complex with the sigma(54)-RNA polymerase holoenzyme. The crystal structure of AMP-AlF(x)*PspF(1-275) provides new information on protein-nucleotide interactions and suggests that the beta and gamma phosphates are more important than the alpha phosphate in terms of sensing nucleotide bound states. In addition, functional data obtained with AMP-AlF(x) establish distinct roles for the conserved catalytic AAA(+) (ATPases associated with various cellular activities) residues, suggesting that AMP-AlF(x) is a powerful new tool to study AAA(+) protein family members and, more generally, Walker motif ATPases.
Abstract: AAA+ proteins (ATPases associated with various cellular activities) contribute to many cellular processes and typically function as higher order oligomers permitting the coordination of nucleotide hydrolysis for functional output, which leads to substrate remodeling. The precise mechanisms that enable the relay of nucleotide hydrolysis to their specific functional outputs are largely unknown. Here we use PspF, a specialized AAA+ protein required for enhancer-dependent transcription activation in Escherichia coli, as a model system to address this question. We demonstrate that a conserved asparagine is involved in internal organization of the oligomeric ring, regulation of ATPase activity by "trans" factors, and optimizing substrate remodeling. We provide evidence that the spatial relationship between the asparagine residue and the Walker B motif is one key element in the conformational signaling pathway that leads to substrate remodeling. Such functional organization most likely applies to other AAA+ proteins, including Ltag (simian virus 40), Rep40 (Adeno-associated virus-2), and p97 (Mus musculus) in which the asparagine to Walker B motif relationship is conserved.
Abstract: Bacterial sigma (sigma) factors confer gene specificity upon the RNA polymerase, the central enzyme that catalyses gene transcription. The binding of the alternative sigma factor sigma(54) confers upon the RNA polymerase special functional and regulatory properties, making it suited for control of several major adaptive responses. Here, we summarize our current understanding of the interactions the sigma(54) factor makes with the bacterial transcription machinery.
Abstract: AAA(+) proteins are ubiquitous mechanochemical ATPases that use energy from ATP hydrolysis to remodel their versatile substrates. The AAA(+) characteristic hexameric ring assemblies raise important questions about if and how six often identical subunits coordinate hydrolysis and associated motions. The PspF AAA(+) domain, PspF(1-275), remodels the bacterial sigma(54)-RNA polymerase to activate transcription. Analysis of ATP substrate inhibition kinetics on ATP hydrolysis in hexameric PspF(1-275) indicates negative homotropic effects between subunits. Functional determinants required for allosteric control identify: (i) an important link between the ATP bound ribose moiety and the SensorII motif that would allow nucleotide-dependent *-helical */beta subdomain dynamics; and (ii) establishes a novel regulatory role for the SensorII helix in PspF, which may apply to other AAA(+) proteins. Consistent with functional data, homotropic control appears to depend on nucleotide state-dependent subdomain angles imposing dynamic symmetry constraints in the AAA(+) ring. Homotropic coordination is functionally important to remodel the sigma(54) promoter. We propose a structural symmetry-based model for homotropic control in the AAA(+) characteristic ring architecture.
Abstract: Regulated gene expression requires control of the transcription machinery, frequently through the establishment of different functional states of the transcribing enzyme RNA polymerase and its attendant activator proteins. In bacteria, major adaptive responses use an enhancer-dependent RNA polymerase, activated for transcription by a class of ATPases that remodel initial promoter complexes to form transcriptionally proficient open promoter complexes. In the present article, we summarize the integrated use of site-specific protein cleavage and DNA cross-linking methods, as well as FRET (fluorescence resonance energy transfer) in combination with X-ray crystallography and cryo-electron microscopy to gain insight into the organization of the enhancer-dependent sigma 54-RNA polymerase and the ATPase-driven activation mechanism.
Abstract: Transcriptional initiation invariably involves the transition from a closed RNA polymerase (RNAP) promoter complex to a transcriptional competent open complex. Activators of the bacterial sigma(54)-RNAP are AAA+ proteins that couple ATP hydrolysis to restructure the sigma(54)-RNAP promoter complex. Structures of the sigma(54) activator PspF AAA+ domain (PspF(1-275)) bound to sigma(54) show two loop structures proximal to sigma(54) as follows: the sigma(54) contacting the GAFTGA loop 1 structure and loop 2 that classifies sigma(54) activators as pre-sensor 1 beta-hairpin AAA+ proteins. We report activities for PspF(1-275) mutated in the AAA+ conserved sensor I threonine/asparagine motif (PspF(1-275)(T148A), PspF(1-275)(N149A), and PspF(1-275)(N149S)) within the second region of homology. We show that sensor I asparagine plays a direct role in ATP hydrolysis. However, low hydrolysis rates are sufficient for functional output in vitro. In contrast, PspF(1-275)(T148A) has severe defects at the distinct step of sigma(54) promoter restructuring. This defect is not because of the failure of PspF(1-275)(T148A) to stably engage with the closed sigma(54) promoter, indicating (i) an important role in ATP hydrolysis-associated motions during energy coupling for remodeling and (ii) distinguishing PspF(1-275)(T148A) from PspF(1-275) variants involved in signaling to the GAFTGA loop 1, which fail to stably engage with the promoter. Activities of loop 2 PspF(1-275) variants are similar to those of PspF(1-275)(T148A) suggesting a functional signaling link between Thr(148) and loop 2. In PspF(1-275) this link relies on the conserved nucleotide state-dependent interaction between the Walker B residue Glu(108) and Thr(148). We propose that hydrolysis is relayed via Thr(148) to loop 2 creating motions that provide mechanical force to the GAFTGA loop 1 that contacts sigma(54).
Abstract: The bacterial enhancer binding proteins (bEBP) are members of the AAA+ protein family and have a highly conserved 'DE' Walker B motif thought to be involved in the catalytic function of the protein with an active role in nucleotide hydrolysis. Based on detailed structural data, we analysed the functionality of the conserved 'DE' Walker B motif of a bEBP model, phage shock protein F (PspF), to investigate the role of these residues in the sigma(54)-dependent transcription activation process. We established their role in the regulation of PspF self-association and in the relay of the ATPase activity to the remodelling of an RNA polymerase.promoter complex (Esigma(54).DNA). Specific substitutions of the conserved glutamate (E) allowed the identification of new functional ATP.bEBP.Esigma(54) complexes which are stable and transcriptionally competent, providing a new tool to study the initial events of the sigma(54)-dependent transcription activation process. In addition, we show the importance of this glutamate residue in sigma(54).DNA conformation sensing, permitting the identification of new intermediate stages within the transcription activation pathway.
Abstract: Initiation of transcription is a major point of transcriptional regulation and invariably involves the transition from a closed to an open RNA polymerase (RNAP) promoter complex. In the case of the sigma(54)-RNAP, this multi step process requires energy, provided by ATP hydrolysis occurring within the AAA+ domain of enhancer binding proteins (EBPs). Typically, EBPs have an N-terminal regulatory domain, a central AAA+ domain that directly contacts sigma(54) and a C-terminal DNA binding domain. The following AAA+ EBP crystal structures have recently become available: heptameric AAA+ domains of NtrC1 and dimeric NtrC1 with its regulatory domain, hexameric AAA+ domains of ZraR with DNA binding domains, apo and nucleotide bound forms of the AAA+ domain of PspF as well as a cryo-EM structure of the AAA+ domain of PspF complexed with sigma(54). These AAA+ domains reveal the structural conservation between EBPs and other AAA+ domains. EBP specific structural features involved in substrate remodelling are located proximal to the pore of the hexameric ring. Parallels with the substrate binding elements near the central pore of other AAA+ members are drawn. We propose a structural model of EBPs in complex with a sigma(54)-RNAP-promoter complex.
Abstract: Control of gene expression is key to development and adaptation. Using purified transcription components from bacteria, we employ structural and functional studies in an integrative manner to elaborate a detailed description of an obligatory step, the accessing of the DNA template, in gene expression. Our work focuses on a specialized molecular machinery that utilizes ATP hydrolysis to initiate DNA opening and permits a description of how the events triggered by ATP hydrolysis within a transcriptional activator can lead to DNA opening and transcription. The bacterial EBPs (enhancer binding proteins) that belong to the AAA(+) (ATPases associated with various cellular activities) protein family remodel the RNAP (RNA polymerase) holoenzyme containing the sigma(54) factor and convert the initial, transcriptionally silent promoter complex into a transcriptionally proficient open complex using transactions that reflect the use of ATP hydrolysis to establish different functional states of the EBP. A molecular switch within the model EBP we study [called PspF (phage shock protein F)] is evident, and functions to control the exposure of a solvent-accessible flexible loop that engages directly with the initial RNAP promoter complex. The sigma(54) factor then controls the conformational changes in the RNAP required to form the open promoter complex.
Abstract: The catalytic AAA+ domain (PspF1-275) of an enhancer-binding protein is necessary and sufficient to contact sigma54-RNA polymerase holoenzyme (Esigma54), remodel it, and in so doing catalyze open promoter complex formation. Whether ATP binding and hydrolysis is coordinated between subunits of PspF and the precise nature of the nucleotide(s) bound to the oligomeric forms responsible for substrate remodeling are unknown. We demonstrate that ADP stimulates the intrinsic ATPase activity of PspF1-275 and propose that this heterogeneous nucleotide occupancy in a PspF1-275 hexamer is functionally important for specific activity. Binding of ADP and ATP triggers the formation of functional PspF1-275 hexamers as shown by a gain of specific activity. Furthermore, ATP concentrations congruent with stoichiometric ATP binding to PspF1-275 inhibit ATP hydrolysis and Esigma54-promoter open complex formation. Demonstration of a heterogeneous nucleotide-bound state of a functional PspF1-275.Esigma54 complex provides clear biochemical evidence for heterogeneous nucleotide occupancy in this AAA+ protein. Based on our data, we propose a stochastic nucleotide binding and a coordinated hydrolysis mechanism in PspF1-275 hexamers.
Abstract: MalT, the dedicated transcriptional activator of the maltose regulon in Escherichia coli, is subject to multiple controls. Maltotriose, the inducer, promotes MalT self-association, a critical step in promoter binding, whereas three proteins acting as negative allosteric effectors (MalK, the ABC-component of the maltodextrin transporter, MalY, and Aes) antagonize maltotriose binding. All of these regulatory signals are integrated by a novel signal transduction module that comprises three out of the four MalT structural domains: DT1, the ATP-binding domain that contains determinants recognized by the negative effectors, DT2, and DT3, the maltotriose-binding domain. For a better insight into the role of DT3 in signal integration, we PCR mutagenized the DT3-encoding region and screened for gain of function mutations in a malK+ strain in the absence of repression by MalY or Aes. Most of the mutations isolated alter one of seven residues that are located in DT3 helices 10 and 11, or in the turn between them and delineate a surface-exposed motif. In vivo and in vitro analyses revealed that the substitutions altering the so-called H10/H11 motif do not affect the ability of MalT to activate transcription or its sensitivity to MalY and Aes, but dramatically decrease its sensitivity to MalK. We propose that MalT/MalK interaction might involve two distinct contact sites on each partner. These sites would be located in DT1 and DT3 of MalT, and in the nucleotide-binding domain and the regulatory domain of MalK. Such a two-point interaction model would explain how the regulatory activity of MalK might be coupled to transport.
Abstract: MalK, the ATP-binding cassette component of the Escherichia coli maltodextrin transporter, has long been known to control negatively the activity of MalT, a transcriptional activator dedicated to the maltose regulon. By using a biochemical approach and the soluble form of MalK as a model substrate, we demonstrate that MalK alone inhibits transcription activation by MalT in a purified transcription system. The inhibitory effect observed in vitro is relieved by maltotriose and by two malT mutations and one malK mutation known to interfere with MalT repression by MalK in vivo. MalK interacts directly with the activator in the absence of maltotriose but not in the presence of maltotriose. Conversely, MalK inhibits maltotriose binding by MalT. Altogether, these data strongly suggest that MalK and maltotriose compete for MalT binding. Part, if not all, of the MalK-binding site is located on DT1, the N-terminal domain of MalT. All of these features indicate that MalK inhibits MalT by the same mechanism as two other proteins, MalY and Aes, that also act as negative effectors of MalT by antagonizing maltotriose binding by MalT. These results offer new insights into the mechanism by which gene regulation can be accomplished by the ATPase component of a bacterial ATP-binding cassette-type importer.
Abstract: MalT, the transcriptional activator of the maltose regulon from Escherichia coli, is the prototype of a new family of transcription factors. Its activity is controlled by multiple regulatory signals. ATP and maltotriose (the inducer) are two effectors of the activator that positively control its multimerization, a critical step in promoter binding. In addition, MalK, the ABC component of the maltodextrin transport system, and the two enzymes MalY and Aes down-regulate MalT activity in vivo. By using a biochemical approach, we demonstrate here that (i) Aes controls MalT activity through direct protein-protein interaction, (ii) Aes competes with maltotriose for MalT binding, (iii) ATP and ADP differentially affect the competition between Aes and the inducer, and (iv) part, if not all, of the Aes binding site is located in DT1, the N-terminal domain of the activator, which also contains the ATP binding site. All of these characteristics point toward an identical mode of action for MalY and Aes. However, we have identified an amino acid substitution in MalT that suppresses MalT inhibition by Aes without interfering with its inhibition by MalY, suggesting that the binding sites of the two inhibitory proteins do not coincide. The differential effects of ATP and ADP on the competition between the inducer and Aes (or MalY) suggest that the ATPase activity displayed by MalT plays a role in the negative control of its activity.
