RESUMO
Transcription is fundamentally noisy, leading to significant heterogeneity across bacterial populations. Noise is often attributed to burstiness, but the underlying mechanisms and their dependence on the mode of promotor regulation remain unclear. Here, we measure E. coli single cell mRNA levels for two stress responses that depend on bacterial sigma factors with different mode of transcription initiation (σ70 and σ54). By fitting a stochastic model to the observed mRNA distributions, we show that the transition from low to high expression of the σ70-controlled stress response is regulated via the burst size, while that of the σ54-controlled stress response is regulated via the burst frequency. Therefore, transcription initiation involving σ54 differs from other bacterial systems, and yields bursting kinetics characteristic of eukaryotic systems.
Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , Trifosfato de Adenosina/química , Teorema de Bayes , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrólise , Cinética , Modelos Genéticos , Regiões Promotoras Genéticas , RNA Polimerase Sigma 54/metabolismo , RNA Mensageiro/metabolismo , Fator sigma/metabolismo , Análise de Célula Única , Processos EstocásticosRESUMO
Bacteria synthesize inorganic polyphosphate (polyP) in response to a variety of different stress conditions. polyP protects bacteria by acting as a protein-stabilizing chaperone, metal chelator, or regulator of protein function, among other mechanisms. However, little is known about how stress signals are transmitted in the cell to lead to increased polyP accumulation. Previous work in the model enterobacterium Escherichia coli has indicated that the RNA polymerase-binding regulatory protein DksA is required for polyP synthesis in response to nutrient limitation stress. In this work, I set out to characterize the role of DksA in polyP regulation in more detail. I found that overexpression of DksA increases cellular polyP content (explaining the long-mysterious phenotype of dksA overexpression rescuing growth of a dnaK mutant at high temperatures) and characterized the roles of known functional residues of DksA in this process, finding that binding to RNA polymerase is required but that none of the other functions of DksA appear to be necessary. Transcriptomics revealed genome-wide transcriptional changes upon nutrient limitation, many of which were affected by DksA, and follow-up experiments identified complex interactions between DksA and the stress-sensing alternative sigma factors FliA, RpoN, and RpoE that impact polyP production, indicating that regulation of polyP synthesis is deeply entwined in the multifactorial stress response network of E. coliIMPORTANCE Inorganic polyphosphate (polyP) is an evolutionarily ancient, widely conserved biopolymer required for stress resistance and pathogenesis in diverse bacteria, but we do not understand how its synthesis is regulated. In this work, I gained new insights into this process by characterizing the role of the transcriptional regulator DksA in polyP regulation in Escherichia coli and identifying previously unknown links between polyP synthesis and the stress-responsive alternative sigma factors FliA, RpoN, and RpoE.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Polifosfatos/metabolismo , RNA Polimerase Sigma 54/metabolismo , Fator sigma/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Ligação Proteica , RNA Polimerase Sigma 54/genética , Fator sigma/genética , Estresse FisiológicoRESUMO
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.
Assuntos
Proteínas AAA/metabolismo , Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteínas AAA/química , Proteínas AAA/genética , Trifosfato de Adenosina/metabolismo , Bactérias/química , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , RNA Polimerase Sigma 54/química , RNA Polimerase Sigma 54/genética , RNA Polimerase Sigma 54/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Bactérias/enzimologia , Bactérias/metabolismo , RNA Polimerase Sigma 54/metabolismo , Iniciação da Transcrição Genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Regiões Promotoras Genéticas , Conformação Proteica , RNA Polimerase Sigma 54/química , RNA Polimerase Sigma 54/genéticaRESUMO
BACKGROUND: Bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important crop diseases in the world. More insights into the mechanistic regulation of bacterial pathogenesis will help us identify novel molecular targets for developing effective disease control strategies. A large flagellar gene cluster is regulated under a three-tiered hierarchy by σ54 factor RpoN2 and its activator FleQ, and σ28 factor FliA. A hypothetical protein gene fliTX is located upstream of rpoN2, however, how it is regulated and how it is related to bacterial behaviors remain to be elucidated. RESULTS: Sequence alignment analysis indicated that FliTX in Xoo is less well conserved compared with FliT proteins in Escherichia coli, Salmonella typhimurium, and Pseudomonas fluorescens. Co-transcription of fliTX with a cytosolic chaperone gene fliS and an atypical PilZ-domain gene flgZ in an operon was up-regulated by RpoN2/FleQ and FliA. Significantly shorter filament length and impaired swimming motility were observed in ∆fliTX compared with those in the wildtype strain. ∆fliTX also demonstrated reduced disease lesion length and in planta growth in rice, attenuated ability of induction of hypersensitive response (HR) in nonhost tobacco, and down-regulation of type III secretion system (T3SS)-related genes. In trans expression of fliTX gene in ∆fliTX restored these phenotypes to near wild-type levels. CONCLUSIONS: This study demonstrates that RpoN2- and FliA-regulated fliTX is indispensible for flagellar motility and virulence and provides more insights into mechanistic regulation of T3SS expression in Xoo.
Assuntos
Proteínas de Bactérias/genética , Flagelos/fisiologia , Regulação Bacteriana da Expressão Gênica , Doenças das Plantas/microbiologia , RNA Polimerase Sigma 54/metabolismo , Fator sigma/metabolismo , Xanthomonas/metabolismo , Xanthomonas/patogenicidade , Proteínas de Bactérias/metabolismo , Flagelos/genética , Oryza/microbiologia , RNA Polimerase Sigma 54/genética , Fator sigma/genética , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Virulência , Xanthomonas/genéticaRESUMO
Clostridium difficile, a major cause of nosocomial diarrhea and pseudomembranous colitis, still poses serious health-care challenges. The expression of its two main virulence factors, TcdA and TcdB, is reportedly repressed by cysteine, but molecular mechanism remains unclear. The cysteine desulfidase CdsB affects the virulence and infection progresses of some bacteria. The C. difficile strain 630 genome encodes a homolog of CdsB, and in the present study, we analyzed its role in C. difficile 630Δerm by constructing an isogenic ClosTron-based cdsB mutant. When C. difficile was cultured in TY broth supplemented with cysteine, the cdsB gene was rapidly induced during the exponential growth phase. The inactivation of cdsB not only affected the resistance of C. difficile to cysteine, but also altered the expression levels of intracellular cysteine-degrading enzymes and the production of hydrogen sulfide. This suggests that C. difficile CdsB is a major inducible cysteine-degrading enzyme. The inactivation of the cdsB gene in C. difficile also removed the cysteine-dependent repression of toxin production, but failed to remove the Na2S-dependent repression, which supports that the cysteine-dependent repression of toxin production is probably attributable to the accumulation of cysteine by-products. We also mapped a δ54 (SigL)-dependent promoter upstream from the cdsB gene, and cdsB expression was not induced in response to cysteine in the cdsR::ermB or sigL::ermB strain. Using a reporter gene fusion analysis, we identified the necessary promoter sequence for cysteine-dependent cdsB expression. Taken together, these results indicate that CdsB is a key inducible cysteine desulfidase in C. difficile which is regulated by δ54 and CdsR in response to cysteine and that cysteine-dependent regulation of toxin production is closely associated with cysteine degradation.
Assuntos
Clostridioides difficile/enzimologia , Clostridioides difficile/genética , Cisteína/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/biossíntese , Toxinas Bacterianas/biossíntese , Sítios de Ligação , Clostridioides difficile/metabolismo , Enterotoxinas/biossíntese , Deleção de Genes , Sulfeto de Hidrogênio/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase Sigma 54/metabolismo , Sulfatos/metabolismoRESUMO
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.
Assuntos
Bactérias/química , Bactérias/enzimologia , RNA Polimerase Sigma 54/química , RNA Polimerase Sigma 54/metabolismo , Transcrição Gênica , Trifosfato de Adenosina/metabolismo , Bactérias/genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , RNA Polimerase Sigma 54/genéticaRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Biologia Molecular/métodos , RNA Polimerase Sigma 54/metabolismo , Transcrição Gênica/fisiologia , Adenosina Trifosfatases/química , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Cromatografia em Camada Fina , Pegada de DNA/métodos , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Técnicas In Vitro , Dados de Sequência Molecular , RNA Polimerase Sigma 54/química , Transativadores/isolamento & purificação , Fatores de Transcrição/isolamento & purificaçãoRESUMO
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.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase Sigma 54/química , Transcrição Gênica , Domínio Catalítico , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/química , Corantes Fluorescentes , Nucleotídeos/metabolismo , Estrutura Terciária de Proteína , RNA Polimerase Sigma 54/metabolismo , Moldes GenéticosRESUMO
The σ(54)-dependent transcription in bacteria requires specific activator proteins, bacterial enhancer binding protein (bEBP), members of the AAA+ (ATPases Associated with various cellular Activities) protein family. The bEBPs usually form oligomers in order to hydrolyze ATP and make open promoter complexes. The bEBP formed by HrpR and HrpS activates transcription from the σ(54)-dependent hrpL promoter responsible for triggering the Type Three Secretion System in Pseudomonas syringae pathovars. Unlike other bEBPs that usually act as homohexamers, HrpR and HrpS operate as a highly co-dependent heterohexameric complex. To understand the organization of the HrpRS complex and the HrpR and HrpS strict co-dependence, we have analyzed the interface between subunits using the random and directed mutagenesis and available crystal structures of several closely related bEBPs. We identified key residues required for the self-association of HrpR (D32, E202 and K235) with HrpS (D32, E200 and K233), showed that the HrpR D32 and HrpS K233 residues form interacting pairs directly involved in an HrpR-HrpS association and that the change in side-chain length at position 233 in HrpS affects self-association and interaction with the HrpR and demonstrated that the HrpS D32, E200 and K233 are not involved in negative regulation imposed by HrpV. We established that the equivalent residues K30, E200 and E234 in a homo-oligomeric bEBP, PspF, are required for the subunit communication and formation of an oligomeric lock that cooperates with the ATP γ-phosphate sensing PspF residue R227, providing insights into their roles in the heteromeric HrpRS co-complex.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Fatores de Transcrição/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Subunidades Proteicas , Pseudomonas syringae/química , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , RNA Polimerase Sigma 54/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Nucleotídeos/metabolismo , RNA Polimerase Sigma 54/metabolismo , Transativadores/metabolismo , Adenosina Trifosfatases/química , Espectrometria de Massas , Modelos Biológicos , Nucleotídeos/química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , RNA Polimerase Sigma 54/química , Transativadores/química , Transcrição GênicaRESUMO
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.
Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Bactérias/química , Proteínas de Ligação a DNA/química , RNA Polimerase Sigma 54/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Bactérias/genética , Bactérias/metabolismo , Berílio/química , Cristalização , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fluoretos/química , Expressão Gênica , Estrutura Terciária de Proteína , RNA Polimerase Sigma 54/genética , RNA Polimerase Sigma 54/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transcrição GênicaRESUMO
It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54-RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase and the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.
Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Moleculares , Nucleotídeos/metabolismo , RNA Polimerase Sigma 54/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Hidrólise , Klebsiella pneumoniae/genética , Ligação Proteica , Estrutura Terciária de Proteína , Sinorhizobium meliloti/genéticaRESUMO
BACKGROUND: Heterologous expression of bacterial biosynthetic gene clusters is currently an indispensable tool for characterizing biosynthetic pathways. Development of an effective, general heterologous expression system that can be applied to bioprospecting from metagenomic DNA will enable the discovery of a wealth of new natural products. METHODOLOGY: We have developed a new Escherichia coli-based heterologous expression system for polyketide biosynthetic gene clusters. We have demonstrated the over-expression of the alternative sigma factor σ(54) directly and positively regulates heterologous expression of the oxytetracycline biosynthetic gene cluster in E. coli. Bioinformatics analysis indicates that σ(54) promoters are present in nearly 70% of polyketide and non-ribosomal peptide biosynthetic pathways. CONCLUSIONS: We have demonstrated a new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where high-level pleiotropic sigma factors from the heterologous host directly and positively regulate transcription of the non-native biosynthetic gene cluster. Our bioinformatics analysis is consistent with the hypothesis that heterologous expression mediated by the alternative sigma factor σ(54) may be a viable method for the production of additional polyketide products.
Assuntos
Vias Biossintéticas , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Policetídeos/metabolismo , RNA Polimerase Sigma 54/metabolismo , Antibacterianos/farmacologia , Sequência de Bases , Benzotiazóis , Diaminas , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Dados de Sequência Molecular , Compostos Orgânicos/metabolismo , Oxitetraciclina/biossíntese , Oxitetraciclina/química , Oxitetraciclina/farmacologia , Peptídeos/metabolismo , Policetídeos/química , Regiões Promotoras Genéticas/genética , Quinolinas , RNA Polimerase Sigma 54/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Bacterial enhancer binding proteins (bEBPs) are a subclass of the AAA(+) (ATPases Associated with various cellular Activities) protein family. They are responsible for σ(54)-dependent transcription activation during infection and function under many stressful growth conditions. The majority of bEBPs are regulated in their formation of ring-shaped hexameric self-assemblies via an amino-terminal domain through its phosphorylation or ligand binding. In contrast, the Escherichia coli phage shock protein F (PspF) is negatively regulated in trans by phage shock protein A (PspA). Up to six PspA subunits suppress PspF hexamer action. Here, we present biochemical evidence that PspA engages across the side of a PspF hexameric ring. We identify three key binding determinants located in a surface-exposed 'W56 loop' of PspF, which form a tightly packed hydrophobic cluster, the 'YLW' patch. We demonstrate the profound impact of the PspF W56 loop residues on ATP hydrolysis, the σ(54) binding loop 1, and the self-association interface. We infer from single-chain studies that for complete PspF inhibition to occur, more than three PspA subunits need to bind a PspF hexamer with at least two binding to adjacent PspF subunits. By structural modelling, we propose that PspA binds to PspF via its first two helical domains. After PspF binding-induced conformational changes, PspA may then share structural similarities with a bEBP regulatory domain.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Transativadores/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico/química , Hidrólise , Modelos Biológicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , RNA Polimerase Sigma 54/metabolismo , Transativadores/químicaRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Bactérias Gram-Negativas/química , RNA Polimerase Sigma 54/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Dimerização , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , RNA Polimerase Sigma 54/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Transativadores/química , Transativadores/metabolismoRESUMO
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.
Assuntos
Proteínas de Escherichia coli/química , RNA Polimerase Sigma 54/química , Transativadores/química , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Sítios de Ligação , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Subunidades Proteicas/química , RNA Polimerase Sigma 54/metabolismo , Transativadores/metabolismo , Ativação TranscricionalRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Lipase/genética , Pseudomonas alcaligenes/genética , RNA Polimerase Sigma 54/genética , Transcrição Gênica/genética , Adenosina Trifosfatases/metabolismo , Ácido Aspártico/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Bacteriana da Expressão Gênica , Lipase/biossíntese , Mutação , Fosforilação , Pseudomonas alcaligenes/enzimologia , Pseudomonas alcaligenes/metabolismo , RNA Polimerase Sigma 54/química , RNA Polimerase Sigma 54/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Alginate overproduction by Pseudomonas aeruginosa, also known as mucoidy, is associated with chronic endobronchial infections in cystic fibrosis. Alginate biosynthesis is initiated by the extracytoplasmic function sigma factor (σ(22); AlgU/AlgT). In the wild-type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered to the cytoplasmic membrane by the anti-sigma factor MucA that inhibits alginate production. One mechanism underlying the conversion to mucoidy is mutation of mucA. However, the mucoid conversion can occur in wt mucA strains via the degradation of MucA by activated intramembrane proteases AlgW and/or MucP. Previously, we reported that the deletion of the sensor kinase KinB in PAO1 induces an AlgW-dependent proteolysis of MucA, resulting in alginate overproduction. This type of mucoid induction requires the alternate sigma factor RpoN (σ(54)). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB rpoN double mutant. In the kinB mutant of PAO1, RpoN controlled the expression of approximately 20% of the genome. In addition to alginate biosynthetic and regulatory genes, KinB and RpoN also control a large number of genes including those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, BALB/c mice exhibited increased survival when challenged with the kinB mutant relative to survival with PAO1 challenge. Together, these data strongly suggest that KinB regulates virulence factors important for the development of acute pneumonia and conversion to mucoidy.