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Isolation and properties of enhancer-bypass mutants of sigma 54
Abstract
The N-terminal activation domain of Escherichia coli sigma 54 was randomly mutated to provide a library of changes that might allow the required enhancer function to be bypassed. Five clones harbouring mutant sigma factors were obtained that exhibited this property in that they enhanced growth under nitrogen-limiting conditions in cells lacking NtrC. DNA sequence analysis located all mutations to four leucines in a small region between amino acids 25 and 31. No mutant sigma factors retained the hydrophobic character of the leucine residues. Mutant sigma factors were shown to transcribe in vitro without the need for enhancer binding activator or ATP hydrolysis, confirming the in vivo phenotype. These and other data suggest that a very small set of leucines is critical for keeping polymerase function in check, allowing high responsiveness to physiological induction via enhancer proteins such as NtrC.
... Based on previous experiments, the N-terminus of sigma 54 is an obvious candidate to contribute to these interactions. Various mutations in the N-terminus lead to altered control of melting (J.T.Wang et al., 1995 Syed and Gralla, 1997; Cannon et al., 1999). N-terminal deletion allows promoter DNA to be melted transiently by the mutant holoenzyme without activator, leading to some unregulated transcription. ...
... We proposed previously that there may be regulatory cooperation between the N-terminus and the conserved –12 region promoter sequences. Part of the basis for this proposal was that unregulated transient melting can be caused by mutations within either the N-terminus of sigma 54 or within the –12 sequences of the promoter (J.T.Wang et al., 1995 Syed and Gralla, 1997; Wang and Gralla, 1998). If this proposal is true then one might see the same altered junction recognition patterns whether it is the sigma 54 that is mutated or the promoter DNA. ...
... The N-terminus of sigma 54 appears to play a central role in this regulatory switch. The N-terminus contains motifs including a leucine patch (Wang et al., 1995; Syed and Gralla, 1997) which are required for regulated melting in vivo and in vitro (Dwight and Gralla, 1990; Wong et al., 1994; J.T.Wang et al., 1997). The N-terminus also contains sequences proposed to mediate the effect of activator (Syed and Gralla, 1998). ...
Results of binding assays using DNA fork junction probes indicate that sigma 54 contains multiple determinants that regulate melting to allow RNA polymerase to remain in closed promoter complexes in order to respond to enhancers. Gel mobility shift studies indicate that the -12 promoter element and parts of sigma 54 act together to form a molecular switch that controls melting. The DNA sequences and the sigma 54 N-terminus help direct polymerase to the location within the -12 promoter element where melting will initiate. However, the fork junction that would lead to melting does not form, due to the action of an inhibitory DNA element. Such unregulated melting is inhibited further by the lack of availability of the single-strand binding elements, which are needed to spread opening from the junction to the transcription start site. Thus, in the absence of looping enhancer protein, proper regulation is maintained as the sigma 54 polymerase remains bound in an inactive state. These complex protein-DNA interactions allow the controls over protein recruitment and DNA melting to be separated, enhancing the diversity of accessible mechanisms of transcription regulation.
... In vitro Transcriptional Run-off Assays: P2 and P3 Promoters are Regulated by RpoN and RpoD Using the Same Initiation Start Site -DNA sequence examination of the promoter region corresponding to the -327 TSS suggested that the transcription initiation could be mediated by RNA polymerase containing either RpoN or housekeeping sigma factor RpoD or subjected to a dual usage. Eσ N cannot transcribe on its own, since it requires ATP-dependent activators like NtrC belonging to the AAA family (41). NtrC is known to act as an enhancer-like protein for many RpoN-regulated by guest on September 18, 2016 http://www.jbc.org/ ...
... NtrC is known to act as an enhancer-like protein for many RpoN-regulated by guest on September 18, 2016 http://www.jbc.org/ Downloaded from promoters (41,42). A consensus matching the RpoN-regulated promoter is located in the -12 and -24 regions and a recognition site for NtrC is also present (Fig. 3). ...
The RpoE sigma factor is essential for the viability of Escherichia coli. RpoE regulates extracytoplasmic functions including lipopolysaccharide (LPS) translocation and some of its non-stoichiometric modifications. Transcription of the rpoE gene is positively autoregulated by EσE and by unknown mechanisms that control the expression of its distally located promoter(s). Mapping of 5′ ends of rpoE mRNA identified five new transcriptional initiation sites (P1 to P5) located distal to EσE-regulated promoter. These promoters are activated in response to unique signals. Out of these P2, P3 and P4 defined major promoters, recognized by RpoN, RpoD and RpoS sigma factors, respectively. Isolation of trans-acting factors, in vitro transcriptional and gel retardation assays revealed that the RpoN-recognized P2 promoter is positively regulated by QseE/F two-component system and NtrC activator, while the RpoD-regulated P3 promoter is positively regulated by Rcs system in response to defects in LPS core biosynthesis, overproduction of certain lipoproteins and by the global regulator CRP. Strains synthesising Kdo2-LA LPS caused up to 7-fold increase in the rpoEP3 activity, which was abrogated in Δ(waaC rcsB). Overexpression of a novel 73 nt sRNA rirA (RfaH interacting RNA) generated by the processing of 5′UTR of the waaQ mRNA induces the rpoEP3 promoter activity concomitant with a decrease in LPS content and defects in the O-antigen incorporation. In the presence of RNA polymerase, RirA binds LPS regulator RfaH known to prevent premature transcriptional termination of waaQ and rfb operons. Excess of RirA could titrate out RfaH causing LPS defects and the activation of rpoE transcription.
... The silencing of unregulated transcription from the optimal binding fork junction has been related to forming a structure that masks the determinants needed for binding the nontemplate single strand. The amino ter-minus of the protein plays a central role (Wang et al. 1995(Wang et al. , 1997Syed and Gralla 1997;Cannon et al. 1999;Guo et al. 1999). Figure 1F shows that the identity of the unpaired −11 nucleotide is not centrally important in this regulation. ...
... This conformational change in the enzyme, however, is not sufficient to complete the connection to the unpaired start site region. This final connection requires changes involving the amino terminus of 54 (Wang et al. 1995;Cannon et al. 1999;Gallegos et al. 1999;Guo et al. 1999); mutation of this region allows partial engagement of the start site even without activator ( Fig. 1; Wang et al. 1995Wang et al. , 1997Syed and Gralla 1997). One clue here is that the activator-dependent establishment of the connected interaction between the fork junction and the adjacent nontemplate strand requires ATP binding but not hydrolysis (Fig. 4). ...
Transcription control at the melting step is not yet understood. Here, band shift, cross-linking, and transcription experiments on diverse DNA probes were used with two bacterial RNA polymerase holoenzymes that differ in how they regulate melting. Data indicated that both ς⁵⁴ and ς⁷⁰ holoenzymes assume a default closed form that cannot establish single-strand binding. Upon activation the enzymes are converted to an open form that can bind simultaneously to the upstream fork junction and to the melted transcription start site. The key difference is that ς⁵⁴imposes tighter regulation by creating a complex molecular switch at −12/−11; the current data show that this switch can be thrown by activator. In this case an ATP-bound enhancer protein causes ς⁵⁴ to alter its cross-linking pattern near −11 and also causes a reorganization of holoenzyme: DNA interactions, detected by electrophoretic mobility-shift assay. At a temperature-dependent ς⁷⁰ promoter, elevated temperature alone can assist in triggering conformational changes that enhance the engagement of single-strand DNA. Thus, the two ς factors modify the same intrinsic opening pathway to create quite different mechanisms of transcriptional regulation.
Keywords
• ς factors
• transcription
• promoter opening
• NtrC
• DNA fork junction
... However, the mutations are within sequences with a predominantly acidic character (Merrick, 1993) that do not appear to contact DNA directly Cannon et al., 1995a). Certain mutants in the amino terminal activation domain of N are reported partially to escape the usual activator requirement for DNA melting and form holoenzymes that weakly melt the DNA unaided (Wang et al., 1995;Wang and Gralla, 1996;Syed and Gralla, 1997). Assuming melting is along the natural activated pathway (differences in the stability of the activated and the activator bypass open complexes exist), N appears to contribute to regulating strand separation (Wang and Gralla, 1996). ...
... Mutations leading to a deficiency in open promoter complex formation but without a severe loss of closed promoter complex formation lie within several different parts of N . These include (i) deletions and substitutions in the amino terminal region I Cannon et al., 1995a;Sasse-Dwight and Gralla, 1990), a region which can also be modified to give a low level of activator-independent transcription (Wang et al., 1995;Syed and Gralla, 1997); and (ii) deletion of residues 293-332 in E. coli N surface-exposed element(s) . The activator DctD can be cross-linked to residue 306 of S. typhimurium N (Lee and Hoover, 1995). ...
The sigmaN RNA polymerase binds promoters in a transcriptionally inactive form. Activation by enhancer binding positive control proteins results in the formation of an open promoter complex. In the closed complex, DNA sequences melted upon activation are close contacted by the sigmaN C-terminal DNA-binding domain. Conserved phenylalanine residues within the DNA-binding domain were mutated to examine their contribution to sigmaN function. Mutants defective in supporting sigmaN-dependent growth and in vivo promoter activation were obtained. The mutant proteins were able to bind promoter DNA and to form an RNA polymerase holoenzyme closed complex in vitro. However, they were defective in response to activator in vitro. They failed in the formation of heparin-stable promoter complexes characteristic of open promoter complexes. The sigmaN mutant forms, displaying good promoter occupancy but poor open complex formation, appear defective for some function of the holoenzyme required after initial promoter recognition. The possibilities that the defect could be located in a DNA contact important for DNA melting or is associated with activator interaction and conformational change in sigmaN are discussed.
... Presumably, R336A would disrupt the interactions that are key to hold RI and ELH in position. T30 mutations are defective in transcription including interacting with activators (32), and single mutations in L25, L26, or L28 can bypass the requirement of activators (33). The structures also explain the σ 54 promoter sequence preference of 5′-−18 TTTTGCA −12 -3′ (NT) (34). ...
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 (σ ⁵⁴ ), 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 σ ⁵⁴ 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 σ ⁵⁴ inhibition while helping to open up DNA, using σ ⁵⁴ amino-terminal peptide as a pry bar.
... Where σ N departs from the structure of σ 70 is in region I, also contained within the N-terminal domain. A 50-residue activation motif is contained in this region that is unique to σ N and is critical for transcription initiation (33)(34)(35). While σ N is functionally similar to all sigma factors (i.e., sigma factors initiate transcription from a specified set of promoters), it achieves this by a mechanism that is unique to the σ N family. ...
Regulation of Escherichia coli Pathogenesis by Alternative Sigma Factor N, Page 1 of 2
Abstract
σN (also σ54) is an alternative sigma factor subunit of the RNA polymerase complex that regulates the expression of genes from many different ontological groups. It is broadly conserved in the Eubacteria with major roles in nitrogen metabolism, membrane biogenesis, and motility. σN is encoded as the first gene of a five-gene operon including rpoN (σN), ptsN , hpf , rapZ, and npr that has been genetically retained among species of Escherichia, Shigella, and Salmonella. In an increasing number of bacteria, σN has been implicated in the control of genes essential to pathogenic behavior, including those involved in adherence, secretion, immune subversion, biofilm formation, toxin production, and resistance to both antimicrobials and biological stressors. For most pathogens how this is achieved is unknown. In enterohemorrhagic Escherichia coli (EHEC) O157, Salmonella enterica, and Borrelia burgdorferi, regulation of virulence by σN requires another alternative sigma factor, σS, yet the model by which σN-σS virulence regulation is predicted to occur is varied in each of these pathogens. In this review, the importance of σN to bacterial pathogenesis is introduced, and common features of σN-dependent virulence regulation discussed. Emphasis is placed on the molecular mechanisms underlying σN virulence regulation in E. coli O157. This includes a review of the structure and function of regulatory pathways connecting σN to virulence expression, predicted input signals for pathway stimulation, and the role for cognate σN activators in initiation of gene systems determining pathogenic behavior.
... Although wildtype PspA-PspF-σ 54 exhibits a linear correlation between ATPase activity and transcriptional activation, the mutated system does not: Zhang et al. (2013) produced PspF variants essentially without ATPase activity yet hyperactive transcriptionally (e.g., variant G58C). Additionally, enhancer-bypass mutations of σ 54 can fully alleviate the requirement for a bEBP (Syed and Gralla, 1997;Chaney and Buck, 1999). Hence, the energy dependence of tran-Coiled-coil mediated regulation of AAA+ proteins 9 scriptional activation in the natural EBP-σ 54 system is likely used for tight transcriptional control (see e.g. ...
Phage shock protein A (PspA) belongs to the highy conserved PspA/IM30 family and is a key component of the stress inducible Psp system in Escherichia coli. One of its central roles is the regulatory interaction with the transcriptional activator of this system, the σ(54) enhancer binding protein PspF, a member of the AAA+ protein family. The PspA/F regulatory system has been intensively studied and serves as a paradigm for AAA+ enzyme regulation by trans-acting factors. However, the molecular mechanism of how exactly PspA controls the activity of PspF and hence σ(54) -dependent expression of the psp genes is still unclear. To approach this question, we identified the minimal PspF-interacting domain of PspA, solved its structure, determined its affinity to PspF and the dissociation kinetics, identified residues that are potentially important for PspF regulation and analyzed effects of their mutation on PspF in vivo and in vitro. Our data indicate that several characteristics of AAA+ regulation in the PspA·F complex resemble those of the AAA+ unfoldase ClpB, with both proteins being regulated by a structurally highly conserved coiled-coil domain. The convergent evolution of both regulatory domains points to a general mechanism to control AAA+ activity for divergent physiological tasks via coiled-coil domains.
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... The glutamine-and leucine-rich Region I interacts with activator ATPases, core RNAP and the í12 promoter sequence, thus posing an energy barrier to spontaneous isomerization of RPc to RPo. Region I mutations and deletion often resulted in activator-bypass phenotypes [12,13]. Sigma54 Region II is dispensable for RNAP isomerisation and interaction with DNA. ...
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.
... In some cases, the C-domain has also been shown to mediate oligomerization required before ATPase activity and thus transcriptional activation (Porter et al., 1993;, a process that is facilitated by binding to the UAS sequences via their carboxy-terminal DNA binding domains (Mettke et al., 1995). The unusual property of the formation of stable closed promoter complexes appears to be imposed by a leucine-rich region within the N-terminal of 54 , which keeps the activity of RNAP in check (Wang et al., 1995;1997a;Syed and Gralla, 1997). Cross-linking studies with DctD have suggested that the activating interaction takes place with both the 54 -and -subunits of E 54 and the conserved C3 region of the regulator (Lee and Hoover, 1995;Wang et al., 1997b). ...
Transcription from the Pseudomonas-derived σ54-dependent Po promoter of the dmp operon is mediated by the aromatic-responsive regulator DmpR. However, physiological control is superimposed on this regulatory system causing silencing of the DmpR-mediated transcriptional response in rich media until the transition between exponential and stationary phase is reached. Here, the positive role of the nutritional alarmone (p)ppGpp in DmpR regulation of the Po promoter has been identified and investigated in vivo. Overproduction of (p)ppGpp in a Pseudomonas reporter system was found to allow an immediate transcriptional response under normally non-permissive conditions. Conversely (p)ppGpp-deficient Escherichia coli strains were found to be severely defective in DmpR-mediated transcription, demonstrating the requirement for this metabolic signal. A subset of mutations in the β, β′ and σ70 subunits of RNA polymerase, which confer prototrophy on ppGpp0E. coli, was also found to restore specific DmpR-mediated transcription from Po, suggesting that the metabolic signal is mediated directly through the σ54-RNA polymerase. These data provide a direct mechanistic link between the physiological status of the cell and expression from σ54 promoters.
... The central part of the protein is involved in RNA polymerase binding, while mutations in the COOH terminus speci®cally affect promoter-DNA interactions (Coppard & Merrick, 1991;Guo & Gralla, 1997;Cannon et al., 1995). The ®rst 50 highly conserved residues at the NH 2 -terminal part play a central role in the response to transcription factors, and some mutations that``bypass'' the requirement of activator proteins to initiate transcription have been mapped to this region (Syed & Gralla, 1997). Thus, this region is the most likely target of the activator proteins. ...
Activation of gene expression relies on direct molecular interactions between the RNA polymerase and transcription factors. Eubacterial enhancer-binding proteins (EBPs) activate transcription by binding to distant sites and, simultaneously, contacting the sigma(54)-holoenzyme form of the RNA polymerase (Esigma(54)). The interaction between the EBP and Esigma(54) is transient, such that it has been difficult to be studied biochemically. Therefore, the details of this molecular recognition event are not known. Genetic and physical evidences suggest that the highly conserved C3 region in the activation domain of the EBP has major determinants for positive control and for the interaction with Esigma(54). To further investigate the target of this region we searched for extragenic suppressors of some C3 region mutant derivatives of NifA. As a first step we mutagenized Klebsiella pneumoniae rpoN, the gene that codes for sigma(54). A mutant allele, rpoN1320, that suppressed two different NifA derivatives was obtained. Immunodetection of sigma(54) and transcriptional initiation studies demonstrated that the cause of the suppression was an enhanced expression of rpoN. A single point mutation was responsible for the phenotype. It mapped at the -10 region of an unidentified promoter, here denominated rpoNp1, and increased its similarity to the consensus. A second upstream promoter, denominated rpoNp2, was also identified. Its -10 region partially overlaps with the -35 region of rpoNp1. Interestingly, the promoter-up -10 mutation in rpoNp1 caused a reduction in the expression from rpoNp2, likely reflecting a stronger occupancy of the former promoter by the RNA polymerase at the expense of the latter. The presence of two overlapping promoters competing for the RNA polymerase implies a complex regulatory pattern that needs elucidation. The fact that increasing the concentration of sigma(54) in the cell can suppress positive control mutants of NifA adds further evidence for their direct interaction in the activation process.
... The conserved N-terminal 50 amino acids (region I) contain an excess of glutamine residues and characteristic regularly repeated leucines and are required for response to activator proteins (14)(15)(16)(17). Interestingly, certain mutations in this domain result in low-level activatorindependent transcription (18,19). The next 50 or so amino acids (region II) are not conserved in sequence or in number, but are generally acidic and for E. coli N contribute to DNA melting (14,20). ...
The alternative bacterial sigmaN RNA polymerase holoenzyme binds promoters as a transcriptionally inactive complex that is activated by enhancer-binding proteins. Little is known about how sigma factors respond to their ligands or how the responses lead to transcription. To examine the liganded state of sigmaN, the assembly of end-labeled Klebsiella pneumoniae sigmaN into holoenzyme, closed promoter complexes, and initiated transcription complexes was analyzed by enzymatic protein footprinting. V8 protease-sensitive sites in free sigmaN were identified in the acidic region II and bordering or within the minimal DNA binding domain. Interaction with core RNA polymerase prevented cleavage at noncontiguous sites in region II and at some DNA binding domain sites, probably resulting from conformational changes. Formation of closed complexes resulted in further protections within the DNA binding domain, suggesting close contact to promoter DNA. Interestingly, residue E36 becomes sensitive to proteolysis in initiated transcription complexes, indicating a conformational change in holoenzyme during initiation. Residue E36 is located adjacent to an element involved in nucleating strand separation and in inhibiting polymerase activity in the absence of activation. The sensitivity of E36 may reflect one or both of these functions. Changing patterns of protease sensitivity strongly indicate that sigmaN can adjust conformation upon interaction with ligands, a property likely important in the dynamics of the protein during transcription initiation.
... Because transcription requires hydrolysis of the – bond, AMP–PNP can be utilized by the RNA polymerase, but not by RcNtrC for an ATPase activity . This analog has been used previously to investigate the ATPase function for activation by enteric NtrC (e.g., Wang et al. 1995; Syed and Gralla 1997). The RcNtrC C7 protein was used because of its high level of activation that is independent of MBP–NtrB and phosphorylation . ...
A commonly accepted view of gene regulation in bacteria that has emerged over the last decade is that promoters are transcriptionally activated by one of two general mechanisms. The major type involves activator proteins that bind to DNA adjacent to where the RNA polymerase (RNAP) holoenzyme binds, usually assisting in recruitment of the RNAP to the promoter. This holoenzyme uses the housekeeping sigma70 or a related factor, which directs the core RNAP to the promoter and assists in melting the DNA near the RNA start site. A second type of mechanism involves the alternative sigma factor (called sigma54 or sigmaN) that directs RNAP to highly conserved promoters. In these cases, an activator protein with an ATPase function oligomerizes at tandem sites far upstream from the promoter. The nitrogen regulatory protein (NtrC) from enteric bacteria has been the model for this family of activators. Activation of the RNAP/sigma54 holoenzyme to form the open complex is mediated by the activator, which is tethered upstream. Hence, this class of protein is sometimes called the enhancer binding protein family or the NtrC class. We describe here a third system that has properties of each of these two types. The NtrC enhancer binding protein from the photosynthetic bacterium, Rhodobacter capsulatus, is shown in vitro to activate the housekeeping RNAP/sigma70 holoenzyme. Transcriptional activation by this NtrC requires ATP binding but not hydrolysis. Oligomerization at distant tandem binding sites on a supercoiled template is also necessary. Mechanistic and evolutionary questions of these systems are discussed.
... Also, because transcriptional activity of s 54 involves numerous unique stereospeci®c interactions, the preservation of transcriptional activity in the single cysteine mutants and the Cys(±)s 54 protein suggests that the conformation of the mutant proteins important for function is not altered signi®cantly. Region I sequences and R336 are involved in preventing unregulated polymerase isomerization (Syed et al., 1997; Syed and Gralla, 1998; Chaney and Buck, 1999). We therefore tested whether the single cysteine s 54 mutants were active for unregulated transcription (also calledàctivator bypass' transcription), i.e. whether they allowed polymerase isomerization in the absence of activator proteins. ...
Two distinct classes of RNA polymerase sigma factors (sigma) exist in bacteria and are largely unrelated in primary amino acid sequence and their modes of transcription activation. Using tethered iron chelate (Fe-BABE) derivatives of the enhancer-dependent sigma(54), we mapped several sites of proximity to the beta and beta' subunits of the core RNA polymerase. Remarkably, most sites localized to those previously identified as close to the enhancer-independent sigma(70) and sigma(38). This indicates a common use of sets of sequences in core for interacting with the two sigma classes. Some sites chosen in sigma(54) for modification with Fe-BABE were positions, which when mutated, deregulate the sigma(54)-holoenzyme and allow activator-independent initiation and holoenzyme isomerization. We infer that these sites in sigma(54) may be involved in interactions with the core that contribute to maintenance of alternative states of the holoenzyme needed for either the stable closed promoter complex conformation or the isomerized holoenzyme conformation associated with the open promoter complex. One site of sigma(54) proximity to the core is apparently not evident with sigma(70), and may represent a specialized interaction.
... The silencing of unregulated transcription from the optimal binding fork junction has been related to forming a structure that masks the determinants needed for binding the nontemplate single strand. The amino terminus of the protein plays a central role (Wang et al. 1995, 1997; Syed and Gralla 1997; Cannon et al. 1999; Guo et al. 1999). Figure 1F shows that the identity of the unpaired −11 nucleotide is not centrally important in this regulation. ...
Transcription control at the melting step is not yet understood. Here, band shift, cross-linking, and transcription experiments on diverse DNA probes were used with two bacterial RNA polymerase holoenzymes that differ in how they regulate melting. Data indicated that both sigma(54) and sigma(70) holoenzymes assume a default closed form that cannot establish single-strand binding. Upon activation the enzymes are converted to an open form that can bind simultaneously to the upstream fork junction and to the melted transcription start site. The key difference is that sigma(54) imposes tighter regulation by creating a complex molecular switch at -12/-11; the current data show that this switch can be thrown by activator. In this case an ATP-bound enhancer protein causes sigma(54) to alter its cross-linking pattern near -11 and also causes a reorganization of holoenzyme: DNA interactions, detected by electrophoretic mobility-shift assay. At a temperature-dependent sigma(70) promoter, elevated temperature alone can assist in triggering conformational changes that enhance the engagement of single-strand DNA. Thus, the two sigma factors modify the same intrinsic opening pathway to create quite different mechanisms of transcriptional regulation.
... The data indicate that there is remarkable selectivity for these two well separated individual residues with regard to the ability to silence transcription in the absence of activator. This selectivity is in marked contrast to the bulk of bypass mutants, which reside in the N terminus; numerous changes throughout the N-terminal 50 amino acids can lead to the bypass phenotype (19,20,22,33). The contrast indicates that the N terminus is a discrete regulatory motif, whereas the C terminus contains scattered residues that contribute to regulation. ...
Twenty-one conserved positively charged and aromatic amino acids between residues 331 and 462 of sigma 54 were changed to
alanine, and the mutant proteins were studied by transcription, band shift analysis, and footprinting in vitro. A small segment corresponding to the rpoN box was found to be most important for binding duplex DNA. Two amino acids, 52
residues apart, were found to be critical for maintaining transcriptional silencing in the absence of activator. These two
activator bypass mutants and several other mutants failed to bind the type of fork junction DNA thought to be required to
maintain silencing. The two bypass mutants showed a binding pattern to DNA probes that was unique, both in comparison to other
C-terminal mutants and to previously known N-terminal bypass mutants. On this basis, a model is proposed for the role of the
C terminus and the N terminus of sigma 54 in enhancer-dependent transcription.
... Several mutants displayed properties in vitro that were not easily reconciled with their in vivo properties (for example, E36G and E325G). Such differences have been observed before (14,15,40,41) and may relate to non-ideal behaviour of the mutant proteins in in vitro assays and issues of protein stability in vivo, although their precise bases remain unknown. Overall, our study of surfaceexposed residues in σ 54 by site-directed mutagenesis guided by previous protease footprinting studies has yielded a number of mutant σ 54 proteins with a rich array of phenotypes and has identified residues and regions in σ 54 of functional and regulatory significance that otherwise would have escaped analysis. ...
Protein footprints of the enhancer-dependent σ54 protein, upon binding the Escherichia coli RNA polymerase core enzyme or upon forming closed promoter complexes, identified surface-exposed residues in σ54 of potential functional importance at the interface between σ54 and core RNA polymerases (RNAP) or DNA. We have now characterised alanine and glycine substitution mutants at several of
these positions. Properties of the mutant σ54s correlate protein footprints to activity. Some mutants show elevated DNA binding suggesting that promoter binding by holoenzyme
may be limited to enable normal functioning. One such mutant (F318A) within the DNA binding domain of σ54 shows a changed interaction with the promoter regulatory region implicated in transcription silencing and fails to silence
transcription in vitro. It appears specifically defective in preferentially binding to a repressive DNA structure believed to restrict RNA polymerase
isomerisation and is largely intact for activator responsiveness. Two mutants, one in the regulatory region I and the other
within core interacting sequences of σ54, failed to stably bind the activator in the presence of ADP‐aluminium fluoride, an analogue of ATP in the transition state
for hydrolysis. Overall, the data presented describe a collection σ54 mutants that have escaped previous analysis and display an array of properties which allows the role of surface-exposed residues
in the regulation of open complex formation and promoter DNA binding to be better understood. Their properties support the
view that the interface between σ54 and core RNAP is functionally specialised.
Gene transcription is central to the development, differentiation, and adaptation of cells. Control of transcription requires the interplay of signaling pathways with the molecular machinery of transcription, the DNA-dependent RNA polymerase (RNAP) enzyme, regulatory proteins that act upon it, and the nucleic acid that is transcribed. The genetic tractability of bacteria, in particular Escherichia coli and Bacillus subtilis, and yeast has allowed rapid progress in elucidating the types of strategy used for the control of gene expression at the level of transcription. The RNAP is evolutionarily conserved in sequence, structure, and function from bacteria to humans. The simple (in terms of subunit composition) bacterial RNAP is an excellent model system to study the control of gene transcription. This chapter also describes the components of such a system and how they interact to allow regulation of RNAP activity at the level of the DNA opening event (i.e., open complex formation) necessary for trancription initiation.
Sigma 54 is a required factor for bacterial RNA polymerase to respond to enhancers and directs a mechanism that is a hybrid between bacterial and eukaryotic transcription. Three pathways were found that bypass the enhancer requirement in vitro. These rely on either deletion of the sigma 54 N terminus or destruction of the DNA consensus -12 promoter recognition element or altering solution conditions to favor transient DNA melting. Each of these allows unstable heparin-sensitive pre-initiation complexes to form that can be driven to transcribe in the absence of both enhancer protein and ATP beta-gamma hydrolysis. These disparate pathways are proposed to have a common basis in that multiple N-terminal contacts may mediate the interactions between the polymerase and the DNA region where melting originates. The results raise possibilities for common features of open complex formation by different RNA polymerases.
Spo0A is the central regulator of commitment to sporulation in Bacillus subtilis. Spo0A is a member of the response regulator family of proteins and both represses and stimulates transcription from promoters when activated. In vivo Spo0A activation takes place by phosphorylation and in vitro activation can be accomplished by phosphorylation or removal of the N-terminal domain of the protein. We have examined the mechanism of Spo0A stimulation of transcription from the promoter of the spoIIG operon. This operon encodes one of the first compartment specific sigma factors whose appearance regulates sporulation development. When activated Spo0A was incubated with RNA polymerase and a DNA fragment containing the spoIIG promoter, bases between -13 and -3, relative to the start site of transcription, were denatured. Addition of activated Spo0A or RNA polymerase alone did not induce denaturation. Heteroduplex templates that contained the nontemplate sequence of the wild-type promoter on both strands between positions -3 and -13 were efficiently transcribed without activated Spo0A. These data suggest that DNA strand separation is a two-step process and that the activation of Spo0A creates a form that interacts with the polymerase to induce the first of the two steps.
Sigma 54 associates with bacterial core RNA polymerase and converts it into an enhancer-responsive enzyme. Deletion of the N-terminal 40 amino acids is known to result in loss of the ability to respond to enhancer binding proteins. In this work PCR mutagenesis and genetic screens were used to identify a small patch, from amino acids 33 to 37, that is required for proper response to activator in vivo. Site-directed single point mutants within this segment were constructed and studied. Two of these were defective in responding to the enhancer binding protein in vitro. The mutants could still direct the polymerase to bind to DNA and initiate transient melting. However, they failed in directing activator-dependent formation of a heparin-stable open complex. Thus, amino acid region 33 to 37 includes critical activation response determinants. This region overlaps the larger leucine patch negative-control region, suggesting that anti-inhibition and positive activation are closely coupled events.
Activation of transcription at sigma 54-dependent bacterial promoters proceeds via a mechanism that is independent of recruitment of RNA polymerase to the promoter, but is instead totally dependent on activator-driven conformational changes in the promoter-bound RNA polymerase. Understanding of the activation mechanism first requires a detailed description of the interactions taking place in the polymerase holoenzyme and closed complex. The interactions of sigma 54 with core RNA polymerase and promoter DNA were investigated using enzymatic and chemical (hydroxyl radical) protease footprinting of sigma. Regions of sigma were identified that are in direct contact with ligands, or whose conformation changes following ligand binding. A comparison of wild-type sigma and a mutant bearing a deletion of conserved Region I, which is required for response to activator proteins and regulated initiation, revealed differences in the protease sensitivity of free sigma indicating that Region I affects sigma conformation. Comparison of the holoenzyme and closed complex hydroxyl radical footprints revealed that residues of wild-type sigma protected by promoter DNA overlap, to a large extent, the residues of Region I-deleted sigma protected by core polymerase. Region I could thus modify DNA-binding by changing conformation of the DNA-binding domain of sigma 54 in a core polymerase-dependent manner. These differences can account for the modified promoter binding of the Region I-deleted sigma holoenzyme observed by DNA footprinting, and are likely of significance to the Region I-dependent activation of transcription.
Transcription from the Pseudomonas-derived sigma 54-dependent Po promoter of the dmp operon is mediated by the aromatic-responsive regulator DmpR. However, physiological control is superimposed on this regulatory system causing silencing of the DmpR-mediated transcriptional response in rich media until the transition between exponential and stationary phase is reached. Here, the positive role of the nutritional alarmone (p)ppGpp in DmpR regulation of the Po promoter has been identified and investigated in vivo. Overproduction of (p)ppGpp in a Pseudomonas reporter system was found to allow an immediate transcriptional response under normally non-permissive conditions. Conversely (p)ppGpp-deficient Escherichia coli strains were found to be severely defective in DmpR-mediated transcription, demonstrating the requirement for this metabolic signal. A subset of mutations in the beta, beta' and sigma 70 subunits of RNA polymerase, which confer prototrophy on ppGpp0 E. coli, was also found to restore specific DmpR-mediated transcription from Po, suggesting that the metabolic signal is mediated directly through the sigma 54-RNA polymerase. These data provide a direct mechanistic link between the physiological status of the cell and expression from sigma 54 promoters.
The conserved amino-terminal region of sigma 54 (Region I) contains sequences that allow response to activator proteins, and inhibit initiation in the absence of activator. Alanine-scanning mutagenesis has been used to systematically define Region I elements that contribute to each of these functions. Amino acid residues from 6 to 50 were substituted with alanine in groups of three consecutive residues, making a total of 15 mutants. Mutants were tested for their ability to mediate activation in vivo, and in vitro, and to support transcription in the absence of activator in vitro. Most mutations located between residues 15 and 47 altered sigma function, while mutations between residues 6 and 14, and 48-50 had little effect. The defective mutants ala 15-17, 42-44, and 45-47 define new amino acids required for normal sigma function. In general, there is an inverse correlation between the levels of activated and activator-independent transcription, suggesting that the two functions are linked. When activated, the defective sigma mutants, except for ala 24-26, formed heparin-resistant open complexes similar to wild-type sigma. Mutant ala 24-26 formed heparin-unstable open complexes, suggesting that this mutation interferes with a different step in the initiation pathway.
Escherichia coli transcription factor sigma 54 contains motifs that resemble closely those used for RNA polymerase II in mammalian cells, including two hydrophobic heptad repeats, a very acidic region and a glutamine-rich region. Triple changes in hydrophobic or multiple changes in acidic residues in Region III are known to severely impair core-binding ability. To investigate whether all the changes in triple mutants are necessary for core binding, site-directed mutagenesis was performed to create single and double mutants in the leucine or isoleucine residues in the heptad repeat in Region III. Single mutants showed no discernible loss of function. Double mutants showed partial protection of the -12 promoter element of the glnAp2 promoter due to the partial loss of their ability to bind core RNA polymerase. These mutations were deleterious to the function of sigma 54, which retained only 30-40% of wild-type mRNA levels. However, double mutants retained nearly normal ability to form open complexes. Two triple mutants created during previous work lost most, if not all, of their ability to bind core RNA polymerase, to protect the -12 promoter element of the glnAp2 promoter and to open the transcription start site. The two triple mutants produced about 20% or less than 10% of the wild-type transcripts from the glnAp2 promoter. These results demonstrate that the hydrophobic heptad repeat in Region III is essential for core RNA polymerase binding. Progressive loss of hydrophobicity of the hydrophobic heptad repeat in Region III of sigma 54 resulted in a progressive loss of core-binding ability, leading to the loss of -12 promoter element recognition and mRNA production.
The bacterial sigma(54) RNA polymerase functions in a transcription activation mechanism that fully relies upon nucleotide hydrolysis by an enhancer binding activator protein to stimulate open complex formation. Here, we describe results of DNA-binding assays used to probe the role of the sigma(54) amino terminal region I in activation. Of the 15 region I alanine substitution mutants assayed, several specifically failed to bind to a DNA structure representing an early conformation in DNA melting. The same mutants are defective in activated transcription and in forming an isomerised sigma-DNA complex on the early opened DNA. The mechanism of activation may therefore require tight binding of sigma(54) to particular early melted DNA structures. Where mutant sigma(54) binding to early melted DNA was detected, activator-dependent isomerisation generally occurred as efficiently as with the wild-type protein, suggesting that certain region I sequences are largely uninvolved in sigma isomerisation. DNA-binding, sigma isomerisation and transcription activation assays allow formulation of a functional map of region I.
The ς54 subunit of the bacterial RNA polymerase requires the action of specialized enhancer-binding activators to initiate transcription.
Here we show that ς54 is able to melt promoter DNA when it is bound to a DNA structure representing the initial nucleation of DNA opening found
in closed complexes. Melting occurs in response to activator in a nucleotide-hydrolyzing reaction and appears to spread downstream
from the nucleation point toward the transcription start site. We show that ς54 contains some weak determinants for DNA melting that are masked by the Region I sequences and some strong ones that require
Region I. It seems that ς54 binds to DNA in a self-inhibited state, and one function of the activator is therefore to promote a conformational change
in ς54 to reveal its DNA-melting activity. Results with the holoenzyme bound to early melted DNA suggest an ordered series of events
in which changes in core to ς54 interactions and ς54-DNA interactions occur in response to activator to allow ς54 isomerization and the holoenzyme to progress from the closed complex to the open complex.
sigma 54, which is encoded by rpoN, is required for a variety of metabolic functions in bacteria including the utilization of alternative carbon and nitrogen sources, nitrogen fixation, and the expression of virulence determinants. Sequence analysis of a 3,020-bp DNA fragment from the plant pathogen Pseudomonas syringae pv. glycinea PG4180 revealed four ORFs designated rpoN, orfA, orfB, and orfC delta, which were related to rpoN and rpoN-associated genes from other microorganisms. The rpoN upstream region in P. syringae contained two overlapping promoters, which may suggest a complex regulatory pattern. This is the first study describing the organization of the rpoN locus in P. syringae.
Transcription of the Ntr regulon is controlled by the two-component system consisting of the response regulator NRI (NtrC) and the kinase/phosphatase NRII (NtrB), which both phosphorylates and dephosphorylates NRI. Even though in vitro transcription from nitrogen-regulated promoters requires phosphorylated NRI, NRII-independent activation of NRI also occurs in vivo. We show here that this activation likely involves acetyl phosphate; it is eliminated by mutations that reduce synthesis of acetyl phosphate and is elevated by a mutation expected to cause accumulation of acetyl phosphate. With purified components, we investigated the mechanism by which acetyl phosphate stimulates glutamine synthetase synthesis. Acetyl phosphate, carbamyl phosphate, and phosphoramidate but not ATP or phosphoenolpyruvate acted as substrates for the autophosphorylation of NRI in vitro. Phosphorylated NRI produced by this mechanism exhibited the properties associated with NRI phosphorylated by NRII, including the activated ATPase activity of the central domain of NRI and the ability to activate transcription from the nitrogen-regulated glutamine synthetase glnAp2 promoter.
The alternative sigma factor sigma 54 of enteric bacteria, or its homologue in other purple bacteria, is required for transcription of genes whose products have diverse physiological roles. Previous studies have indicated that sigma 54 confers on core RNA polymerase the ability to recognize a specific class of promoters but not the ability to isomerize from closed to open complexes. Isomerization requires ATP and one member of a family of activator proteins, it being different activator proteins that allow this form of polymerase to respond to different physiological signals. We have developed a strategy for overproducing and purifying sigma 54 from Salmonella typhimurium and have studied several biochemical properties of reconstituted sigma 54-holoenzyme. The initial binding constant KB for the formation of closed complexes between this holoenzyme and the ginA promoter in our transcription buffer is approximately 3 x 10(8) M-1, which was determined from DNaseI protection assays at 37 degrees C. After the formation of open complexes, several properties of sigma 54-holoenzyme appear to be similar to those of sigma 70-holoenzyme. We have determined the complete nucleotide sequence of the gene encoding sigma 54 (ntrA) in Salmonella.
The protein nitrogen regulator I (NRI)-phosphate is known to activate the initiation of transcription of the Escherichia coli glnA gene. This activation is facilitated by the binding of the protein to NRI-specific sites located upstream of the sigma 54-dependent glnA promoter. To determine whether binding of NRI-phosphate to upstream sites is sufficient for activation, we placed several promoters not normally activated by NRI-phosphate downstream of NRI binding sites and measured activation in intact cells and in an in vitro transcription system. We found that the sigma 70-dependent lac promoter was not activated, that the sigma 54-dependent Klebsiella pneumoniae nifH promoter was weakly activated, and that a nifH promoter altered in the RNA polymerase binding site was almost as well activated as the glnA promoter. We conclude that the sensitivity of the susceptible promoter depends on the presence of NRI binding sites, but that the presence of bound NRI-phosphate upstream of a promoter is not sufficient for activation of transcription by RNA polymerase. This activation is determined by the structure of the RNA polymerase binding site. We suggest that sigma 54-but not sigma 70-dependent promoters are susceptible to activation by NRI-phosphate and that the nucleotide sequence of the sigma 54-RNA polymerase binding site is an important determinant of the efficiency of activation.
In vivo "footprints" of the glnA regulatory region under activating conditions demonstrate that the three most upstream activator sequences bind the protein NRI in the cell. Together, protections at these sites span six of seven consecutive major grooves and lie on the same helix face. E sigma 54 protects two major grooves of DNA approximately 60 base pairs downstream at the glnAp2 promoter and primarily on the opposite helix face. Experiments using potassium permanganate to probe open complex formation in vivo demonstrate that NRI is absolutely required for E sigma 54 to open the promoter DNA. Together, the dimethyl sulfate and permanganate studies verify [Reitzer, L. J., Bueno, R., Cheng, W. D., Abrams, S. A., Rothstein, D. M., Hunt, T. P., Tyler, B. & Magasanik, B. (1987) J. Bacteriol. 169, 4279-4284] that E sigma 54 occupies the glnAp2 promoter in a closed complex in vivo even in the presence of excess nitrogen and the absence of NRI. Furthermore, the slow step in transcriptional activation is shown to be an NRI-dependent conformational change in the downstream promoter DNA, which results in DNA melting. These observations place interesting restrictions on models describing the mechanism by which NRI activates transcription from glnAp2 at a distance.
In enteric bacteria the products of two nitrogen regulatory genes, ntrA and ntrC, activate transcription of glnA, the structural gene encoding glutamine synthetase, both in vivo and in vitro. The ntrC product (gpntrC) is a DNA-binding protein, which binds to five sites in the glnA promoter-regulatory region and appears to activate transcription initiation. Using as an assay the stimulation of glnA transcription in a coupled in vitro transcription-translation system, we have partially purified the ntrA gene product (gpntrA). The following evidence is consistent with the view that gpntrA is a sigma subunit for RNA polymerase: (i) The gpntrA activity copurifies with the sigma 70 holoenzyme (E sigma 70) and core (E) forms of RNA polymerase through several steps but can be separated from them by chromatography on heparin agarose. (ii) After further purification by molecular sieve chromatography, the partially purified gpntrA fraction allows transcription of glnA from the same startpoint used in vivo; transcription is dependent on gpntrC and on added E. The gpntrA fraction does not allow transcription from promoters that we have used as controls, including lacUV5. E sigma 70 has the reverse specificity.
The nac gene of Klebsiella aerogenes encodes a bifunctional transcription factor that activates or represses the expression of several operons under conditions of nitrogen limitation. In experiments with purified components, transcription from the nac promoter was initiated by sigma 54 RNA polymerase and was activated by the phosphorylated form of nitrogen regulator I (NRI) (NtrC). The activation of the nac promoter required a higher concentration of NRI approximately P than did the activation of the Escherichia coli glnAp2 promoter, and both the promoter and upstream enhancer element contributed to this difference. The nac promoter had a lower affinity for sigma 54 RNA polymerase than did glnAp2, and uninitiated competitor-resistant transcription complexes formed at the nac promoter decayed to competitor-sensitive complexes at a greater rate than did similar complexes formed at the glnAp2 promoter. The nac enhancer, consisting of a single high-affinity NRI-binding site and an adjacent site with low affinity for NRI, was less efficient in stimulating transcription than was the glnA enhancer, which consists of two adjacent high-affinity NRI-binding sites. When these binding sites were exchanged, transcription from the nac promoter was increased and transcription from the glnAp2 promoter was decreased at low concentrations of NRI approximately P. Another indication of the difference in the efficiency of these enhancers is that although activation of a nac promoter construct containing the glnA enhancer was relatively insensitive to subtle alterations in the position of these sites relative to the position of the promoter, activation of the natural nac promoter or a nac promoter construct containing only a single high-affinity NRI approximately P binding site was strongly affected by subtle alterations in the position of the NRI approximately P binding site(s), indicating a face-of-the-helix dependency for activation.
Transcription initiation at the σ54-dependent glnAp2 promoter was studied to follow the state of polymerase as RNA synthesis begins. σ54 polymerase begins transcription in abortive
cycling mode, i.e. after the first bond is made, approximately 75% of the time the short RNA is aborted and synthesis must be restarted. Polymerase
is capable of abortive initiation until it reaches a position beyond +3 and before +7, at which stage polymerase is released
from its promoter contacts and an elongation complex is formed. Initial elongation is accompanied by two transcription bubbles,
one moving with the polymerase and the other remaining at the transcription start site. The σ54-associated polymerase shows
an earlier and more efficient transition out of abortive initiation mode than prior studies of σ70-associated polymerase.
Single and multiple point mutations were introduced to change the 12 glutamine residues within a 37-amino acid region of sigma 54. Multiple changes are shown to be required in order to interfere significantly with the function of this protein which is associated with enhancer-dependent bacterial transcription. Mutation of the central 4 glutamines leads to the production of less m-RNA, caused by an inability to fully open the promoter start site. DNA binding, however, is normal. Mutation of 4 other adjacent glutamines causes the promoter start site to open more readily than wild type, although this enhanced opening is not accompanied by more mRNA. The enhanced DNA melting is not caused by enhanced promoter binding, as indicated by normal protection of the polymerase-bound promoter against dimethyl sulfate attack. The results suggest that multiple glutamines play a role in transducing the melting signal from the enhancer protein to the polymerase.
In vitro transcription, DNase I footprinting, and abortive initiation assays were used to characterize transcription using mutant
forms of sigma 54 shown previously to bypass certain enhancer requirements in vitro. The holoenzymes containing these sigma mutants produce low levels of open complexes at both the glnAp2 and glnHp2 promoters. The open complexes are unusual in that they are destroyed by heparin. Enhancer protein and ATP convert them into
a stable heparin-resistant state. The enhancer response occurs over a similar range of NtrC concentration as occurs with the
wild-type holoenzyme, indicating that the activation determinants have been largely preserved within these mutants. One-round
transcription assays show that the mutant holoenzymes can be driven to transcribe both promoters without NtrC. The unstable
opening induced by these mutations apparently serves as a conduit that can shuttle templates into transcriptionally competent
complexes. The results lead to a model in which activation occurs in two steps. First, the enhancer complex overcomes an inhibitory
effect of the sigma 54 leucine patch and unlocks the melting activity of the holoenzyme. Second, different sigma 54 determinants
are used to drive stabilization of the open complexes, allowing the full transcription potential to be realized.
The alternative sigma factor sigma 54 is required for transcription of nitrogen fixation genes in Klebsiella pneumoniae and other diazotrophs. The nif genes, and other E sigma 54-dependent genes whose products are necessary for a wide range of processes, are postively regulated. A unifying model that is well supported by studies on nif and other nitrogen-regulated (ntr) genes includes the central tenet that sigma 54 confers upon core RNA polymerase the ability to recognize and bind specific promoter sequences, but not the ability to isomerize to the open complex without assistance from the appropriate activator protein. Direct physical evidence for formation of an activator-independent complex between E sigma 54 and the NifA-dependent K. pneumoniae nifH and nifU promoters has, to date, been lacking. Using purified components we have now demonstrated formation of the closed complex at these promoters, indicating that it is an intermediate along the pathway to open complex formation. The closed complex was not detected when conserved features of the promoter were altered by mutation, nor was its stability increased when integration host factor protein was bound adjacent to the E sigma 54 recognition sequence.
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The regulatory regions for 119 Escherichia coli promoters have been analyzed, and the locations of the regulatory sites have been cataloged. The following observations emerge. (i) More than 95% of promoters are coregulated with at least one other promoter. (ii) Virtually all sigma 70 promoters contain at least one regulatory site in a proximal position, touching at least position -65 with respect to the start point of transcription. There are not yet clear examples of upstream regulation in the absence of a proximal site. (iii) Operators within regulons appear in very variable proximal positions. By contrast, the proximal activation sites of regulons are much more fixed. (iv) There is a forbidden zone for activation elements downstream from approximately position -20 with respect to the start of transcription. By contrast, operators can occur throughout the proximal region. When activation elements appear in the forbidden zone, they repress. These latter examples usually involve autoregulation. (v) Approximately 40% of repressible promoters contain operator duplications. These occur either in certain regulons where duplication appears to be a requirement for repressor action or in promoters subject to complex regulation. (vi) Remote operator duplications occur in approximately 10% of repressible promoters. They generally appear when a multiple promoter region is coregulated by cyclic AMP receptor protein. (vii) Sigma 54 promoters do not require proximal or precisely positioned activator elements and are not generally subject to negative regulation. Rationales are presented for all of the above observations.
E. coli sigma 54 protein confers on promoters containing its recognition sequence the ability to be activated from distant DNA sites. Its functional domains include two leucine zipper motifs, an acidic region, and a glutamine-rich domain. Several domains were disrupted and the assembly of mutant transcription complexes was probed in vivo by footprinting. Promoter recognition was seen to depend on a C-terminal region containing a prokaryotic helix-turn-helix motif. Within the resulting stable closed complex, two leucine zipper motifs assist in positioning the sigma 54 polymerase near the DNA region that must be melted upon activation. Finally, DNA opening depends on the sigma 54 acid domain. The uncoupling of promoter recognition from DNA melting, mediated by the unusual domain structure of this prokaryotic protein, may be responsible for sigma 54,s ability to mediate activation from distant sites.
The nitrogen regulatory (NtrC) protein of enteric bacteria, which binds to sites that have the properties of transcriptional
enhancers, is known to activate transcription by a form of RNA polymerase that contains the NtrA protein (sigma 54) as sigma
factor (referred to as sigma 54-holoenzyme). In the presence of adenosine triphosphate, the NtrC protein catalyzes isomerization
of closed recognition complexes between sigma 54-holoenzyme and the glnA promoter to open complexes in which DNA in the region
of the transcription start site is locally denatured. NtrC is not required subsequently for maintenance of open complexes
or initiation of transcription.
The fdhF gene, encoding the selenopolypeptide of formate dehydrogenase (FDHH), has a -12/-24 nif-type consensus promoter. A cis-acting DNA element, which is required for the regulation of the promoter by formate under anaerobic conditions, has been identified. This regulatory sequence of about 25 bp in length is located 110 bp upstream of the transcription start site. By analysing a variety of mutant constructs in this region (5' deletions, internal deletions and point mutations) we were able to identify a hexanucleotide sequence -GTCACG-, which is important for the formate regulation of the fdhF promoter. The data also indicate that this element has many of the properties characteristic of eukaryotic enhancers.
We have shown that the purified glnF (ntrA) product of Escherichia coli binds to core RNA polymerase. Together these proteins initiated transcription at the nitrogen-regulated promoter glnAp2 on a supercoiled template. The initiation of transcription at glnAp2 on a linear template required in addition NRI, the product of glnG (ntrC), and NRII2302, the product of a mutant allele of glnL (ntrB). These results identify the glnF product as a new sigma factor specifically required for the transcription of nitrogen-regulated and of nitrogen-fixation promoters. We propose rpoN as the proper designation for glnF, and sigma 60 for its product. Our results indicate that sigma 60 RNA polymerase recognizes the nitrogen-regulated/nitrogen-fixation promoter consensus sequence C-T-G-G-Y-A-Y-R-N4-T-T-G-C-A. Initiation of transcription in the intact cell appears to require in addition the active form of NRI, the product of glnG. Conversion of NRI to its active form is apparently brought about by NRII, the product of glnL, in response to nitrogen deprivation.
Transcription of the Escherichia coli glnALG operon, whose products are glutamine synthetase and the regulatory proteins NRII and NRI, is activated by nitrogen deprivation. Initiation of transcription at the nitrogen-regulated promoter glnAp2 requires sigma 60, the product of rpoN (glnF, ntrA), and NRI, the product of glnG (ntrC). We have now shown that the ability of this promoter to be activated by a low intracellular concentration of NRI depends on two binding sites for NRI located approximately 110 and 140 bp, respectively, upstream of the start of transcription. Moving these binding sites more than 1000 bp does not diminish the ability of NRI to stimulate transcription at glnAp2. Thus, the NRI binding sites resemble enhancers in eukaryotic cells.
The initiation of transcription from the nitrogen-regulated promoter glnAp2 requires RNA polymerase containing sigma 54, the transcriptional activator NRI, and the protein kinase NRII, responsible for the conversion of NRI to the active NRI-phosphate. NRI-phosphate does not increase the ability of sigma 54-containing RNA polymerase to bind to the promoter, but rather stimulates the conversion of an initial promoter:polymerase complex to the transcriptionally active open complex. The presence on the DNA template of high-affinity binding sites for NRI/NRI-phosphate, normally located 130 and 100 bp upstream of the site of transcription initiation, results in a 4- to 5-fold lowering of the concentration of NRI required for the formation of the open complex. These high-affinity NRI binding sites facilitate open complex formation when they are moved to positions 700 bp further upstream or 950 bp downstream of glnAp2 on linear DNA templates.
The protein sigma 54 associates with Escherichia coli core RNA polymerase to form a holoenzyme that binds promoters but is inactive in the absence of enhancer activation. Here, mutants of sigma 54 enabled polymerases to transcribe without enhancer protein and adenosine triphosphate. The mutations are in leucines within the NH2-terminal glutamine-rich domain of sigma 54. Multiple leucine substitutions mimicked the effect of enhancer protein, which suggests that the enhancer protein functions to disrupt a leucine patch. The results indicate that sigma 54 acts both as an inhibitor of polymerase activity and as a receptor that interacts with enhancer protein to overcome this inhibition, and that these two activities jointly confer enhancer responsiveness.
sigma 54 is the promoter recognition subunit of the form of bacterial RNA polymerase that transcribes from promoters with enhancer elements. DNase footprinting experiments show that sigma 54 is attached selectively to the template strand, which must be single-stranded for transcription initiation. sigma 54 remains bound at the promoter after core polymerase begins elongation, in contrast to the well-established sigma 70-holoenzyme transcription cycle. Permanganate footprinting experiments show that the bound sigma 54 and the elongating core RNA polymerase downstream of it are each associated with a single-strand DNA region. Template commitment assays show that the promoter-bound sigma 54 must be reconfigured before reinitiation of transcription can occur. This unexpected pathway raises interesting possibilities for transcriptional regulation, especially with regard to control at the level of reinitiation.
Sigma 54 is a minor bacterial sigma factor that is not a member of the sigma 70 family of proteins but binds the same core RNA polymerase. Previously, we identified a region of sigma 54 that is important for binding core polymerase. In this work, PCR mutagenesis was used to identify specific amino acids important for this binding. The results show that important residues are clustered most closely in a short sequence that was previously speculated to be potentially homologous to a sequence in sigma 70. The mutagenesis also identifies important residues in the flanking hydrophobic-acidic region of sigma 54, which is absent in sigma 70. Overall, the data indicate that sigma 54 binds core polymerase through a sequence homologous to that of sigma 70 but in addition uses unique motifs to modify this interaction.
To activate transcription of the glnA gene, the dimeric NTRC protein (nitrogen regulatory protein C) of enteric bacteria binds to an enhancer located approximately 100 bp upstream of the promoter. The enhancer is composed of two binding sites for NTRC that are three turns of the DNA helix apart. One role of the enhancer is to tether NTRC in high local concentration near the promoter to allow for its frequent interaction with sigma 54 holoenzyme by DNA looping. We have found that a second role of the enhancer is to ensure oligomerization of NTRC into a complex of at least two dimers that is required for transcriptional activation. Formation of this complex is greatly facilitated by a protein-protein interaction between NTRC dimers that is increased when the protein is phosphorylated.
Bacteria synthesize a number of different sigma factors which allow the co-ordinate expression of groups of genes owing to the ability of sigma to confer promoter-specific transcription initiation on RNA polymerase. In nearly all cases these sigmas belong to a single family of proteins which appear to be related structurally and functionally to the major Escherichia coli sigma factor, sigma 70. A clear exception is the sigma factor sigma 54 (sigma N), encoded by rpoN, which represents a second family of sigmas that is widely distributed in prokaryotes. Studies of sigma 54 (sigma N) have demonstrated that this sigma is quite distinct both structurally and functionally from the sigma 70 family and the mode of transcription initiation which it mediates may have more in common with that found in eukaryotes than that which occurs with sigma 70 and its relatives.
Transcription of many nitrogen-regulated (Ntr) genes requires the phosphorylated form of nitrogen regulator I (NRI, or NtrC), which binds to sites that are analogous to eukaryotic enhancers. A highly conserved regulatory domain contains the site of phosphorylation and controls the function of NRI. We analyzed the effects of substitutions in highly conserved residues that are part of the active site of phosphorylation of NRI in Escherichia coli. Fourteen substitutions of aspartate 54, the site of phosphorylation, impaired the response to nitrogen deprivation. Only one of these variants, NRI D-54-->E (NRI-D54E), could significantly stimulate transcription from glnAp2, the major promoter of the glnALG operon. Cells with this variant grew with arginine as a nitrogen source. Experiments with purified components showed that unphosphorylated NRI-D54E stimulated transcription. In contrast, substitutions at aspartate 11 were not as deleterious as those at aspartate 54. Finally, we showed that NRI-K103R, in which arginine replaces the absolutely conserved lysine, is functionally active and efficiently phosphorylated. This substitution appears to stabilize the phosphoaspartate of NRI. The differences between our results and those from study of homologous proteins suggest that there may be significant differences in the way highly conserved residues participate in the transition to the activated state.
Escherichia coli sigma 54 was analyzed by making a series of 16 internal deletions within its gene and analyzing the properties of the mutant proteins. All of the mutant proteins except one were strongly defective in a growth test that relied on sigma 54 function. Additional assays were applied to determine the causes of these defects. The assays monitored the following properties: the level of protein expression; ability to bind to the -24 promoter element of the glnAP2 promoter in vivo; the ability to bind to the -12 promoter element in vivo; ability to melt the promoter start site in vivo; ability to bind the Rhizobium meliloti nifH promoter in vitro; and the ability to form a sigma 54-core RNA polymerase complex (E sigma 54 holoenzyme) in vitro. The analysis shows a modular structure in that certain regions of the protein predominate in contributing to each of these properties. A large carboxyl region of the protein is essential for promoter binding. A smaller amino-terminal segment is essential for DNA melting. An element essential for the forming the E sigma 54 holoenzyme lies between these two regions. None of these domains resemble those of sigma 70 and this difference is discussed in view of the different transcription mechanisms directed by the two proteins.
sigma 54 is a rare bacterial protein that substitutes for sigma 70 in the case of Escherichia coli genes transcribed by certain activators with enhancer protein-like properties. It contains a strongly acidic region of previously unknown function. Gel mobility-shift assays using sigma 54 deletion mutants show that this region is essential for sigma 54 to bind the core RNA polymerase and recruit it to the promoter. Multiple-point mutational analysis shows that the acidic amino acids and overlapping periodic hydrophobic amino acids are necessary for this binding. Related sequences are not found within the core binding determinant of sigma 70, which binds the same core RNA polymerase. This comparison suggests that the core RNA polymerase interacts differently with the two sigma factors, likely contributing to the critical differences in transcription mechanism in the two cases.
In order to assess the role of leucine repeat motifs within bacterial protein sigma 54, a series of point mutants were introduced into the many leucine residues near the N terminus. Functional assays in vivo showed that the leucine residues that comprise the previously identified heptad repeat motif are selectively important for function. These heptad leucine residues are critical for mRNA production and also for recognition of the -12 promoter element. An internal proline substitution destroys the function of the heptad repeat region, suggesting a possible alpha-helical structure. Mutants with changes in the distal part of this N-terminal region show the interesting property of allowing nearly full levels of open complex formation, while nonetheless reducing the level of mRNA transcripts produced. All of the above-mentioned properties differ from those exhibited by mutating the interdigitated glutamine residues, which were previously found to be closely involved in the DNA melting reaction. The collection of data suggests that the N-terminal region contains overlapping functional motifs, hydrophobic heptad and glutamine-rich, which together appear to constitute the activation domain of sigma 54.
The 2.6 A crystal structure of a fragment of the sigma 70 promoter specificity subunit of E. coli RNA polymerase is described. Residues involved in core RNA polymerase binding lie on one face of the structure. On the opposite face, aligned along one helix, are exposed residues that interact with the -10 consensus promoter element (the Pribnow box), including four aromatic residues involved in promoter melting. The structure suggests one way in which DNA interactions may be inhibited in the absence of RNA polymerase and provides a framework for the interpretation of a large number of genetic and biochemical analyses.