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At least 109 records · Page 6Linked to original sources
PubMed1989

Characterization of the Escherichia coli transcription factor sigma 70: localization of a region involved in the interaction with core RNA polymerase.

A set of internal deletions and frame-shift mutations was made in the structural gene for the major sigma factor of Escherichia coli RNA polymerase (sigma 70). The truncated proteins from these various mutants were examined to determine if they retained the ability to bind core RNA polymerase. Two assays were used to determine core-binding activity. Gel filtration was used to separate free sigma 70 from sigma 70 bound to core polymerase. Immunoprecipitation of polymerase using an anti-alpha-subunit monoclonal antibody was also used to determine if the various truncated proteins were bound to core. Results from these experiments indicate core-binding activity is retained when large portions of the sigma 70 protein are deleted. Deletion of a region in the central portion of the protein caused a large decrease in core-binding activity. The results suggest that the region spanning amino acids 361-390 is important for efficient core-binding activity. Sequence comparison of various sigma factors shows highly conserved amino acids in this region. A synthetic peptide having the sequence of amino acids 361-390 was synthesized and examined for the ability to bind core RNA polymerase.

Amino Acid Sequence↗
PubMed2002

Crystal structure of the Bacillus stearothermophilus anti-sigma factor SpoIIAB with the sporulation sigma factor sigmaF.

Cell type-specific transcription during Bacillus sporulation is established by sigmaF. SpoIIAB is an anti-sigma that binds and negatively regulates sigmaF, as well as a serine kinase that phosphorylates and inactivates the anti-anti-sigma SpoIIAA. The crystal structure of sigmaF bound to the SpoIIAB dimer in the low-affinity, ADP form has been determined at 2.9 A resolution. SpoIIAB adopts the GHKL superfamily fold of ATPases and histidine kinases. A domain of sigmaF contacts both SpoIIAB monomers, while 80% of the sigma factor is disordered. The interaction occludes an RNA polymerase binding surface of sigmaF, explaining the SpoIIAB anti-sigma activity. The structure also explains the specificity of SpoIIAB for its target sigma factors and, in combination with genetic and biochemical data, provides insight into the mechanism of SpoIIAA anti-anti-sigma activity.

Adenosine Triphosphate↗
PubMed2002

The extracytoplasmic function (ECF) sigma factors.

Bacterial sigma (sigma) factors are an essential component of RNA polymerase and determine promoter selectivity. The substitution of one sigma factor for another can redirect some or all of the RNA polymerase in a cell to activate the transcription of genes that would otherwise be silent. As a class, alternative sigma factors play key roles in coordinating gene transcription during various stress responses and during morphological development. The extracytoplasmic function (ECF) sigma factors are small regulatory proteins that are quite divergent in sequence relative to most other sigma factors. Many bacteria, particularly those with more complex genomes, contain multiple ECF sigma factors and these regulators often outnumber all other types of sigma factor combined. Examples include Bacillus subtilis (7 ECF sigma factors), Mycobacterium tuberculosis (10), Caulobacter crescentus (13), Pseudomonas aeruginosa (approximately 19), and Streptomyces coelicolor (approximately 50). The roles and mechanisms of regulation for these various ECF sigma factors are largely unknown, but significant progress has been made in selected systems. As a general trend, most ECF sigma factors are cotranscribed with one or more negative regulators. Often, these include a transmembrane protein functioning as an anti-sigma factor that binds, and inhibits, the cognate sigma factor. Upon receiving a stimulus from the environment, the sigma factor is released and can bind to RNA polymerase to stimulate transcription. In many ways, these anti-sigma:sigma pairs are analogous to the more familiar two-component regulatory systems consisting of a transmembrane histidine protein kinase and a DNA-binding response regulator. Both are mechanisms of coordinating a cytoplasmic transcriptional response to signals perceived by protein domains external to the cell membrane. Here, I review current knowledge of some of the better characterized ECF sigma factors, discuss the variety of experimental approaches that have proven productive in defining the roles of ECF sigma factors, and present some unifying themes that are beginning to emerge as more systems are studied.

Bacillus subtilis↗
PubMed1995

Sigma factor and sporulation genes in Clostridium.

The genus Clostridium, represented by Gram-positive, anaerobic, spore-forming bacteria, is well known for its clinical importance and considerable biotechnological potential. Recently, evidence for a functional role of the transcription factors sigma A, sigma E, sigma G, and sigma K in this genus was provided by cloning and sequencing these genes from C. acetobutylicum. In C. kluyveri, a partially sequenced open reading frame was found to encode the N terminus of the putative sigma factor L with significant similarity to members of the sigma 54 family. The identification of sequences with high similarity to the Bacillus sigma F (C. acetobutylicum), sigma H (several clostridial species), and sigma D (C. thermocellum)-controlled consensus promoters renders the existence of these transcription factors in clostridia very likely. These data are in agreement with information obtained by RNA transcript mapping (sigma A, sigma H), heterologous DNA hybridization (sigma D, sigma H), and immuno characterization of purified proteins (sigma A) from various clostridial species. Thus, the picture emerges that a fundamental similarity exists at the genetic level between the regulation of various cellular responses, in particular sporulation, in the genera Bacillus and Clostridium. The different induction patterns of sporulation in Bacillus spp. (nutrient starvation) and many clostridial species (cessation of growth or exposure to oxygen in the presence of excess nutrients) are most interestingly not reflected in the general regulatory features of this developmental process.

Amino Acid Sequence↗
PubMed1999

Obg, an essential GTP binding protein of Bacillus subtilis, is necessary for stress activation of transcription factor sigma(B).

sigma(B), the general stress response sigma factor of Bacillus subtilis, is activated when intracellular ATP levels fall or the bacterium experiences environmental stress. Stress activates sigma(B) by means of a collection of regulatory kinases and phosphatases (the Rsb proteins), which catalyze the release of sigma(B) from an anti-sigma factor inhibitor. By using the yeast dihybrid selection system to identify B. subtilis proteins that could interact with Rsb proteins and act as mediators of stress signaling, we isolated the GTP binding protein, Obg, as an interactor with several of these regulators (RsbT, RsbW, and RsbX). B. subtilis depleted of Obg no longer activated sigma(B) in response to environmental stress, but it retained the ability to activate sigma(B) by the ATP responsive pathway. Stress pathway components activated sigma(B) in the absence of Obg if the pathway's most upstream effector (RsbT) was synthesized in excess to the inhibitor (RsbS) from which it is normally released after stress. Thus, the Rsb proteins can function in the absence of Obg but fail to be triggered by stress. The data demonstrate that Obg, or a process under its control, is necessary to induce the stress-dependent activation of sigma(B) and suggest that Obg may directly communicate with one or more sigma(B) regulators.

Adenosine Triphosphate↗
PubMed1989

Tandem genes encoding sigma-factors for consecutive steps of development in Bacillus subtilis.

During sporulation, Bacillus subtilis undergoes successive morphological changes that can be arrested at various stages by mutations in many genes. One of these, spoIIGB, encodes a transcriptional factor, sigma E, which is necessary to proceed beyond stage II and to differentiate the cell in two compartments, the forespore and the mother cell. Mutations were introduced in an open reading frame located immediately downstream of spoIIGB. They block sporulation at stage III and define a new gene, spoIIIG, encoding a 260-amino-acid polypeptide highly similar to bacterial sigma-factors. A promoter was identified in the spoIIGB-spoIIIG interval by transcriptional fusion to lacZ. It is turned on 1 hr after the start of sigma E synthesis and is specifically activated in the forespore. The tandemly arranged spoIIGB and spoIIIG genes appear to encode homologous proteins that modulate transcription in a sequential fashion during sporulation.

Amino Acid Sequence↗
PubMed1999

Involvement of ppGpp, ribosome modulation factor, and stationary phase-specific sigma factor sigma(S) in the decrease in cell viability caused by spermidine.

Accumulation of spermidine in Escherichia coli causes a decrease in cell viability at the late stationary phase of cell growth. The mechanism underlying this effect has been studied. Spermidine accumulation caused an increase in the level of ppGpp and a decrease in ribosome modulation factor (RMF) and stationary phase-specific sigma factor sigma(S), both of which are believed to be involved in cell viability. Transformation of E. coli with the gene for stringent factor, which synthesizes ppGpp, also caused a significant decrease in the levels of RMF and sigma(S) factor and a decrease in cell viability. The results strongly suggest that the accumulation of ppGpp is also involved in the decrease in cell viability and that the sigma(S) factor assists the function of RMF in cell viability.

Bacterial Proteins↗
PubMed2002

Activity of the Streptomyces coelicolor stress-response sigma factor sigmaH is regulated by an anti-sigma factor.

The alternative sigma factor sigmaH has been shown to play an important role in stress response and morphological differentiation in Streptomyces coelicolor. Its gene, sigH, is located in an operon with the gene encoding proposed anti-sigma factor UshX, and one of the promoters directing expression of the operon is dependent upon sigH. To clarify the function of S. coelicolor UshX, both the sigmaH and UshX proteins were overproduced in Escherichia coli and purified. In an in vitro transcription assay, sigmaH, after complementation with S. coelicolor core RNA polymerase, was able to recognize the sigH-dependent promoter, sigH-P2. This transcription was inhibited by UshX, if it was incubated with sigmaH prior to the addition of the core RNA polymerase. When sigmaH and UshX were incubated and electrophoresed through non-denaturing polyacrylamide gels, they formed a specific complex. These results showed that UshX is a specific anti-sigma factor for sigmaH, and the S. coelicolor sigH operon is directly autoregulated.

Bacterial Proteins↗
PubMed1993

Heterogeneity of the principal sigma factor in Escherichia coli: the rpoS gene product, sigma 38, is a second principal sigma factor of RNA polymerase in stationary-phase Escherichia coli.

The rpoS gene of Escherichia coli encodes a putative RNA polymerase sigma factor that is considered to be the central regulator of gene expression in stationary phase. The gene product (sigma 38) was overproduced using the cloned rpoS gene and purified to homogeneity. Reconstituted RNA polymerase holoenzyme (E sigma 38) was found to recognize in vitro a number of typical sigma 70-type promoters, including the lacUV5 and trp promoters. Some, however, were recognized exclusively or preferentially by E sigma 70, whereas at least one, fic, was favored by E sigma 38. Thus E. coli promoters can be classified into three groups: the first group is recognized by E sigma 70 and E sigma 38, but the second and third groups are recognized substantially by either E sigma 70 or E sigma 38 alone. In contrast to other minor sigma factors, sigma 38 shares a set of amino acid sequences common among the principal sigma factors of eubacteria and is therefore a member of the RpoD-related protein family. The intracellular level of sigma 38 was demonstrated to increase in vivo upon entry into stationary phase. These results together indicate that sigma 38 is a second principal sigma factor in stationary-phase E. coli.

Bacterial Proteins↗
PubMed2004

Interaction of Bacillus subtilis extracytoplasmic function (ECF) sigma factors with the N-terminal regions of their potential anti-sigma factors.

Extracytoplasmic function (ECF) sigma factors constitute a diverse family of proteins, within the class of the sigma 70 subunit of RNA polymerase. Most members of the family studied to date are known to regulate gene expression in response to stress conditions. The Bacillus subtilis genome encodes at least 17 distinct sigma factors, seven of which are members of the ECF subfamily. Among these, five sigma factors, namely SigV, SigW, SigX, SigY and SigM, are encoded by the first genes of the cognate sigma operons. Disruption or repressed expression of the downstream gene(s) resulted in transcriptional activation of the cognate sigma operon. Moreover, in vivo protein-protein interaction analyses by yeast two-hybrid experiments indicated that these immediate downstream gene products bind the cognate ECF sigma factor, suggesting that they function as anti-sigma factors by capturing sigma factor on the inner surface of the cytoplasmic membrane. Interaction with other sigma factors was not observed. The results presented here also show that these anti-sigma factors interact with ECF sigma factors through their N-terminal region, implying that the N-terminal domain resides inside the cytoplasmic membrane.

Bacillus subtilis↗
PubMed2000

The anti-sigma factor SpoIIAB forms a 2:1 complex with sigma(F), contacting multiple conserved regions of the sigma factor.

The developmental regulatory protein sigma(F) of Bacillus subtilis, a member of the sigma(70)-family of bacterial RNA polymerase sigma factors, is negatively regulated by the anti-sigma factor SpoIIAB, which binds to sigma(F), sequestering it in an inactive complex. SpoIIAB binding to sigma(F) is strongly stimulated by ATP. Here, we use a combination of gel filtration chromatography, dynamic light-scattering, analytical ultracentrifugation, limited proteolysis with N-terminal sequencing and electrospray mass spectrometry, and deletion analysis to probe the SpoIIAB-sigma(F) complex. The studies were facilitated by investigating the homologs from Bacillus stearothermophilus as well as co-expression of the proteins in Escherichia coli, allowing purification of large quantities of the in vivo assembled complex. We determined the stoichiometry of the complex to be SpoIIAB(2):sigma(F)(1). Alone, sigma(F) is rapidly degraded by the protease trypsin. In the complex with SpoIIAB, however, sigma(F) is remarkably resistant to proteolysis. Analysis of the protease cleavage data indicates the anti-sigma binds sigma(F) through contacts with mutliple conserved regions of the sigma factor, supporting previous findings based on genetic data.

Bacillus subtilis↗
PubMed2003

Expression of primary sigma factor (PSF) and PSF-like sigma factors in the cyanobacterium Synechocystis sp. strain PCC 6803.

Large amounts of sigA mRNA, encoding the primary sigma factor (PSF) in Synechocystis sp. strain PCC 6803, accumulated under standard growth conditions, while stress conditions like heat or high salinity led to a rapid decrease in sigA mRNA content. The sigB, sigC, sigD, and sigE genes, encoding PSF-like sigma factors, were under strict physiological control.

Bacterial Proteins↗
PubMed1997

Characterization of three cDNA species encoding plastid RNA polymerase sigma factors in Arabidopsis thaliana: evidence for the sigma factor heterogeneity in higher plant plastids.

By database search analysis, we identified three Arabidopsis EST (Expression Sequence Tag) entries having similarity to eubacterial RNA polymerase sigma factors. cDNA clones corresponding to these partial sequences were isolated, and the complete nucleotide sequences were determined. All three sequences encode proteins highly homologous to cyanobacterial and plastid sigma factors, and the gene products have N-terminal extensions which are assumed to function as plastid-targeting transit peptides. Thus we have concluded that the gene products are RNA polymerase sigma factors of plastids, and named sigA, sigB and sigC, respectively. Expression of these genes was analyzed by RNA gel-blot analysis and shown to be induced by illumination after a short-term dark adaptation. This strongly suggests that light regulation of the nuclear encoded sigma factor genes is involved in light-dependent activation of plastid promoters.

Amino Acid Sequence↗
PubMed1990

Control of developmental transcription factor sigma F by sporulation regulatory proteins SpoIIAA and SpoIIAB in Bacillus subtilis.

The sporulation operon spoIIA of Bacillus subtilis consists of three cistrons called spoIIAA, spoIIAB, and spoIIAC. Little is known about the function of spoIIAA and spoIIAB, but spoIIAC encodes a sigma factor called sigma F, which is capable of directing the transcription in vitro of genes that are expressed in the forespore chamber of the developing sporangium. We now report that the products of the spoIIA operon constitute a regulatory system in which SpoIIAA is an antagonist of SpoIIAB (or otherwise counteracts the effect of SpoIIAB) and SpoIIAB is, in turn, an antagonist of SpoIIAC (sigma F). This conclusion is based on the observations that (i) overexpression of spoIIAB inhibits sigma F-directed gene expression, (ii) a mutation in spoIIAB stimulates sigma F-directed gene expression, (iii) a mutation in spoIIAA blocks sigma F-directed gene expression, and (iv) a mutation in spoIIAB relieves the block in sigma F-directed gene expression caused by a mutation in spoIIAA. The SpoIIAA/SpoIIAB/SpoIIAC regulatory system could play a role in controlling the timing of sigma F-directed gene expression and/or could be responsible for restricting sigma F-directed gene expression to the forespore chamber of the sporangium.

Bacillus subtilis↗
PubMed1991

Post-transcriptional control of a sporulation regulatory gene encoding transcription factor sigma H in Bacillus subtilis.

The transcriptional regulatory gene spoOH encodes an RNA polymerase sigma factor called sigma H that directs gene expression at an early stage of sporulation in the Gram-positive bacterium Bacillus subtilis. We now report that conditions that induce sporulation cause a rapid increase in the cellular concentration of sigma H. This increase could account for the stimulated transcription of certain sigma H-controlled genes at the onset of sporulation. Experiments in which the expression of spoOH was monitored by use of a spoOH-lacZ fusion and in which expression of spoOH was artificially manipulated by use of an isopropyl-beta-D-thiogalacto-side-inducible promoter indicate that sporulation-induced increases in the amount of sigma H are not controlled at the level of the transcription of its structural gene. Rather, we infer the existence of post-transcriptional control mechanisms that govern sigma H levels, and we present evidence suggesting that increases in the amount of sigma H at the start of sporulation are due to increased translation or stability of the spoOH mRNA and, to a lesser extent, decreased turnover of spoOH protein.

Adenosine↗
PubMed2001

Loss of ribosomal protein L11 blocks stress activation of the Bacillus subtilis transcription factor sigma(B).

sigma(B), the general stress response sigma factor of Bacillus subtilis, is activated when the cell's energy levels decline or the bacterium is exposed to environmental stress (e.g., heat shock, ethanol). Physical stress activates sigma(B) through a collection of regulatory kinases and phosphatases (the Rsb proteins) which catalyze the release of sigma(B) from an anti-sigma(B) factor inhibitor. The means by which diverse stresses communicate with the Rsb proteins is unknown; however, a role for the ribosome in this process was suggested when several of the upstream members of the sigma(B) stress activation cascade (RsbR, -S, and -T) were found to cofractionate with ribosomes in crude B. subtilis extracts. We now present evidence for the involvement of a ribosome-mediated process in the stress activation of sigma(B). B. subtilis strains resistant to the antibiotic thiostrepton, due to the loss of ribosomal protein L11 (RplK), were found to be blocked in the stress activation of sigma(B). Neither the energy-responsive activation of sigma(B) nor stress-dependent chaperone gene induction (a sigma(B)-independent stress response) was inhibited by the loss of L11. The Rsb proteins required for stress activation of sigma(B) are shown to be active in the RplK(-) strain but fail to be triggered by stress. The data demonstrate that the B. subtilis ribosomes provide an essential input for the stress activation of sigma(B) and suggest that the ribosomes may themselves be the sensors for stress in this system.

Bacillus subtilis↗
PubMed2001

Characterization of two genes, Sig1 and Sig2, encoding distinct plastid sigma factors(1) in the moss Physcomitrella patens: phylogenetic relationships to plastid sigma factors in higher plants.

We isolated the cDNA for a sigma factor from the moss Physcomitrella patens, which possesses unusually large N-terminal extension and the conserved subdomains 1.2-4.2. Phylogenetic analyses indicated that this novel sigma factor and PpSIG1*(2), a plastid sigma factor previously identified from Physcomitrella, were classified into SigA and SigB groups, two major classes of higher plant plastid sigma factors, respectively. According to the nomenclature recently proposed, we renamed PpSIG1* into PpSIG2, and named the novel sigma factor PpSIG1. A transient expression assay using a green fluorescent protein showed that the N-terminal region of PpSIG1 acts as a chloroplast-targeting signal. Reverse transcription-PCR experiments showed that light induces the expression of the Sig1 and Sig2 genes encoding PpSIG1 and PpSIG2, respectively. Thus, PpSIG1 and PpSIG2 are likely plastid sigma factors regulating plastid gene expression in response to light signals.

Amino Acid Sequence↗