Article

A fork junction DNA-protein switch that controls promoter melting by the bacterial enhancer-dependent sigma factor

August 1999
The EMBO Journal 18(13):3736-45

August 1999
18(13):3736-45

DOI:10.1093/emboj/18.13.3736

Source
PubMed

Authors:

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.

Promoter opening by sigma 54 and sigma 70 RNA polymerases: sigma factor-directed alterations in the mechanism and tightness of control

Article

Sep 2000
GENE DEV

Yuli Guo

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

In vitro roles of invariant helix-turn-helix motif residue R383 in sigma54 (sigmaN)

Article

Full-text available

Mar 2001
NUCLEIC ACIDS RES

Ramesh Wigneshweraraj

In vitro DNA-binding and transcription properties of σ54 proteins with the invariant Arg383 in the putative helix–turn–helix motif of the DNA-binding domain substituted by lysine or alanine are described. We show that R383 contributes to maintaining stable holoenzyme–promoter complexes in which limited DNA opening downstream of the –12 GC element has occurred. Unlike wild-type σ54, holoenzymes assembled with the R383A or R383K mutants could not form activator-independent, heparin-stable complexes on heteroduplex Sinorhizobium meliloti nifH DNA mismatched next to the GC. Using longer sequences of heteroduplex DNA, heparin-stable complexes formed with the R383K and, to a lesser extent, R383A mutant holoenzymes, but only when the activator and a hydrolysable nucleotide was added and the DNA was opened to include the –1 site. Although R383 appears inessential for polymerase isomerisation, it makes a significant contribution to maintaining the holoenzyme in a stable complex when melting is initiating next to the GC element. Strikingly, Cys383-tethered FeBABE footprinting of promoter DNA strongly suggests that R383 is not proximal to promoter DNA in the closed complex. This indicates that R383 is not part of the regulatory centre in the σ54 holoenzyme, which includes the –12 promoter region elements. R383 contributes to several properties, including core RNA polymerase binding and to the in vivo stability of σ54.

Conservation of sigma-core RNA polymerase proximity relationships between the enhancer-independent and enhancer-dependent sigma classes

Article

Full-text available

Jul 2000

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.

Isomerization of a binary ??-promoter DNA complex by transcription activators

Article

Full-text available

Aug 2000
Nat Struct Biol

Multisubunit RNA polymerases are targets of sophisticated signal transduction pathways that link environmental or temporal cues to changes in gene expression. Here we show that the sigma 54 protein (sigma54), responsible for promoter specific binding by bacterial RNA polymerase, undergoes a nucleotide hydrolysis dependent isomerization on DNA. Changes in protein structure are evident. The isomerization has all the known requirements of sigma 54-dependent transcription, including a dependence on enhancer binding activator proteins and occurs independently of the core RNA polymerase. We suggest that activator driven changes in sigma54 conformation trigger the conversion of a transcriptionally silent RNA polymerase conformation to one able to interact productively with template DNA. Our results illustrate the types of changes that must occur for multisubunit complexes to manipulate DNA, and show that transcription activators can remodel key nucleoprotein structures to achieve direct activation of transcription.

Promoter opening by sigma(54) and sigma(70) RNA polymerases: sigma factor-directed alterations in the mechanism and tightness of control

Article

Oct 2000
GENE DEV

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.

In vitro roles of invariant helix-turn-helix motif residue R383 in σ54 (σN)

Article

Full-text available

Apr 2001
NUCLEIC ACIDS RES

In vitro DNA-binding and transcription properties of sigma(54) proteins with the invariant Arg383 in the putative helix-turn-helix motif of the DNA-binding domain substituted by lysine or alanine are described. We show that R383 contributes to maintaining stable holoenzyme-promoter complexes in which limited DNA opening downstream of the -12 GC element has occurred. Unlike wild-type sigma(54), holoenzymes assembled with the R383A or R383K mutants could not form activator-independent, heparin-stable complexes on heteroduplex Sinorhizobium meliloti nifH DNA mismatched next to the GC. Using longer sequences of heteroduplex DNA, heparin-stable complexes formed with the R383K and, to a lesser extent, R383A mutant holoenzymes, but only when the activator and a hydrolysable nucleotide was added and the DNA was opened to include the -1 site. Although R383 appears inessential for polymerase isomerisation, it makes a significant contribution to maintaining the holoenzyme in a stable complex when melting is initiating next to the GC element. Strikingly, Cys383-tethered FeBABE footprinting of promoter DNA strongly suggests that R383 is not proximal to promoter DNA in the closed complex. This indicates that R383 is not part of the regulatory centre in the sigma(54) holoenzyme, which includes the -12 promoter region elements. R383 contributes to several properties, including core RNA polymerase binding and to the in vivo stability of sigma(54).

Interactions of regulated and deregulated forms of the σ54 holoenzyme with heteroduplex promoter DNA

Article

Full-text available

Mar 2002
NUCLEIC ACIDS RES

The bacterial σ54 RNA polymerase holoenzyme binds to promoters as a stable closed complex that is silent for transcription unless acted upon by an enhancer-bound activator protein. Using DNA binding and transcription assays the ability of the enhancer-dependent σ54 holoenzyme to interact with promoter DNA containing various regions of heteroduplex from –12 to –1 was assessed. Different DNA regions important for stabilising σ54 holoenzyme–promoter interactions, destabilising binding, limiting template utilisation in activator-dependent transcription and for stable binding of a deregulated form of the holoenzyme lacking σ54 Region I were identified. It appears that homoduplex structures are required for early events in σ54 holoenzyme promoter binding and that disruption of a repressive fork junction structure only modestly deregulates transcription. DNA opening from –5 to –1 appears important for stable engagement of the holoenzyme following activation. The regulatory Region I of σ54 was shown to be involved in interactions with the sequences in the –5 to –1 area.

Nucleotide-dependent Triggering of RNA Polymerase-DNA Interactions by an AAA Regulator of Transcription

Article

Full-text available

Jun 2003

Enhancer-dependent activator proteins, which act upon the bacterial RNA polymerase containing the σ54 promoter specificity factor, belong to the AAA superfamily of ATPases. Activator-σ54 contact is required for the σ54-RNAP to isomerize and engage the DNA template for transcription. How ATP hydrolysis is used to trigger changes in σ54-RNA polymerase and promoter DNA that lead to DNA opening is poorly understood. Here, band shift and footprinting assays were used to investigate the DNA binding activities of σ54 and σ54-RNA polymerase in the presence of the activator protein PspF bound to poorly hydrolysable analogues of ATP and the ATP hydrolysis transition-state analogue ADP·AlFx. Results show that different nucleotide-bound forms of PspF can change the interactions between σ54, σ54-RNA polymerase, and a DNA fork junction structure present within closed promoter complexes. This provides evidence that in the activation transduction pathway, several functional states of the activator, prior to ATP hydrolysis, can serve to alter the fork junction binding activity of σ54 and σ54-RNA polymerase that precede full DNA opening. A sequential set of nucleotide-dependent transitions in σ54-RNA polymerase promoter complexes needed for productive open complex formation may therefore depend upon different nucleotide-bound forms of the activator.

Nucleotide-dependent interactions between a fork junction-RNA polymerase complex and an AAA+ transcriptional activator protein

Article

Full-text available

Feb 2004
NUCLEIC ACIDS RES

Enhancer-dependent transcriptional activators that act upon the σ54 bacterial RNA polymerase holoenzyme belong to the extensive AAA+ superfamily of mechanochemical ATPases. Formation and collapse of the transition state for ATP hydrolysis engenders direct interactions between AAA+ activators and the σ54 factor, required for RNA polymerase isomerization. A DNA fork junction structure present within closed complexes serves as a nucleation point for the DNA melting seen in open promoter complexes and restricts spontaneous activator-independent RNA polymerase isomerization. We now provide physical evidence showing that the ADP·AlFx bound form of the AAA+ domain of the transcriptional activator protein PspF changes interactions between σ54-RNA polymerase and a DNA fork junction structure present in the closed promoter complex. The results suggest that one functional state of the nucleotide-bound activator serves to alter DNA binding by σ54 and σ54-RNA polymerase and appears to drive events that precede DNA opening. Clear evidence for a DNA-interacting activity in the AAA+ domain of PspF was obtained, suggesting that PspF may make a direct contact to the DNA component of a basal promoter complex to promote changes in σ54-RNA polymerase–DNA interactions that favour open complex formation. We also provide evidence for two distinct closed promoter complexes with differing stabilities.

A general mechanism for transcription bubble nucleation in bacteria

Article

Full-text available

Mar 2023
P NATL ACAD SCI USA

Bacterial transcription initiation requires σ factors for nucleation of the transcription bubble. The canonical housekeeping σ factor, σ70, nucleates DNA melting via recognition of conserved bases of the promoter -10 motif, which are unstacked and captured in pockets of σ70. By contrast, the mechanism of transcription bubble nucleation and formation during the unrelated σN-mediated transcription initiation is poorly understood. Herein, we combine structural and biochemical approaches to establish that σN, like σ70, captures a flipped, unstacked base in a pocket formed between its N-terminal region I (RI) and extra-long helix features. Strikingly, RI inserts into the nascent bubble to stabilize the nucleated bubble prior to engagement of the obligate ATPase activator. Our data suggest a general paradigm of transcription initiation that requires σ factors to nucleate an early melted intermediate prior to productive RNA synthesis.

Crystal structure of Aquifex aeolicus σ(N) bound to promoter DNA and the structure of σ(N)-holoenzyme

Article

Feb 2017
P NATL ACAD SCI USA

Significance The bacterial σ factors confer promoter specificity to the RNA polymerase (RNAP). One σ factor, σ N , is unique in its structure and functional mechanism, forming transcriptionally inactive promoter complexes with RNAP that require activation by specialized ATPases. The structural basis for σ N function is of great interest but poorly understood. Here, we determined an X-ray crystal structure of a σ N fragment bound to promoter DNA, revealing the molecular details of promoter recognition by σ N . Moreover, the new structure allowed us to build and refine a corrected σ N -holoenzyme (σ N /RNAP complex) model using previously published X-ray data. This work overall provides a solid structural framework with which to address further the poorly understood mechanism of activator function in ATP hydrolysis-dependent promoter opening.

Single amino acid substitution mutants of Klebsiella pneumoniae sigma54 defective in transcription

Article

Nov 2000
NUCLEIC ACIDS RES

Melinda Pitt

Transcription initiation by the σ54 RNA polymerase requires specialised activators and their associated nucleoside triphosphate hydrolysis. To explore the roles of σ54 in initiation we used random mutagenesis of rpoN and an in vivo activity screen to isolate functionally altered σ54 proteins. Five defective mutants, each with a different single amino acid substitution, were obtained. Three failed in transcription after forming a closed complex. One such mutant mapped to regulatory Region I of σ54, the other two to Region III. The Region I mutant allowed transcription independently of activator and showed reduced activator-dependent σ54 isomerisation. The two Region III mutants displayed altered behaviour in a σ54 isomerisation assay and one failed to stably bind early melted DNA as the holoenzyme; they may contribute to a communication pathway linking changes in σ to open complex formation. Two further Region III mutants showed gross defects in overall DNA binding. For one, sufficient residual DNA binding activity remained to allow us to demonstrate that other activities were largely unaffected. Changes in DNA binding preferences and core polymerase-dependent properties were evident amongst the mutants.

Mechanistic studies on transcription activation via DNA looping in a prokaryotic promoter-enhancer system

Article

Jan 2004

Sabine Katja Vogel

In this thesis, transcription activation was studied in a prokaryotic promoter-enhancer system. It comprises the E. coli RNA polymerase, which is associated with the alternative sigma factor sigma54 (RNAP-sigma54), and the transcription activator protein NtrC (nitrogen regulatory protein C), which binds to a remote enhancer region to the promoter. Enhancer-bound NtrC contacts the RNA polymerase at the promoter by means of DNA looping (closed complex) and induces DNA melting of the promoter DNA by RNAP-sigma54 (open complex). The following aspects of this process were studied: (1) Three different sigma54-specific promoter sequences were analyzed in binding studies and in in vitro transcription experiments. These promoters were known to have different overall promoter strength as determined by in vivo expression and DNA footprinting studies. Since initiation of transcription comprises different subsequent steps (promoter-binding by RNAP-sigma54, isomerization to the open complex and formation of a stable elongation complex) it was still unclear, which is the rate limiting step of the total reaction. For the glnAp2 and nifL promoters, the promoter-binding by RNAP-sigma54 was rate limiting. In contrast, for the nifH promoter with a high affinity to RNAP-sigma54 but with a low in vivo expression level, the DNA melting step determined the overall speed of the transcription initiation reaction. (2) By scanning force microscopy it was determined that promoter-bound RNAP-sigma54 bends the DNA and is for this reason preferably localized in the end-loop of a supercoiled DNA, since the DNA is more bent in this region. The localization in the end-loop facilitates the interaction between NtrC and RNAP-sigma54 in spite of the low flexibility of the intervening DNA. (3) The interaction between sigma54 and NtrC was studied in an ATPase assay and in gel shift experiments. It was shown that sigma54 has no effect on the ATPase activity of NtrC under the experimental conditions whereas enhancer-binding of NtrC strongly stimulates the ATPase activity by facilitating the oligomerization of NtrC. Binding studies that were performed by analytical ultracentrifugation and gel shift experiments have also shown that sigma54 alone only weakly bind sthe promoter DNA in contrast to the RNAP-sigma54 holoenzyme. This supports the idea, that the sigma factor acts by recognizing the promoter sequence whereas the RNAP holoenzyme provides the binding energy for high affinity promoter binding. (4) In vitro transcription experiments showed that NtrC activates with different efficiency and can even act as a repressor depending on the position, number and arrangement of its binding sites. Certain combinations of weak and strong NtrC binding sites were shown to activate transcription from the promoter at very different concentrations of NtrC. This enables a regulation of transcription in dependence of the NtrC concentration. From these results, a model of RNAP-sigma54-NtrC-mediated transcription activation was developed: Accordingly, the proximal NtrC sites very close to the promoter facilitate the interaction between activator and RNA polymerase in a loop complex at low NtrC concentrations, whereas at higher concentrations the transcriptional activation is limited to a maximum level. In this case, a NtrC species is formed, which can interact with the RNAP-sigma54 without DNA looping. In dieser Arbeit wurde die Aktivierung der Transkription in einem prokaryotischen Promotor-Enhancer-System untersucht. Es besteht aus der E. coli RNA-Polymerase, die mit der alternativen Sigma-Untereinheit sigma54 assoziiert ist (RNAP-sigma54) und dem Transkriptions-Aktivatorprotein NtrC (Nitrogen regulatory protein C), das vom Promotor entfernt an die Enhancer-Region bindet. NtrC am Enhancer kontaktiert unter Schleifenbildung der DNA die RNA-Polymerase am Promotor (geschlossener Komplex) und induziert das Aufschmelzen der Promotor-DNA durch RNAP-sigma54 (offener Komplex). Folgende Aspekte dieser Reaktion wurden untersucht: (1) In Bindungsstudien und in in vitro Transkriptionsexperimenten wurden drei verschiedene sigma54-spezifischen Promotorsequenzen analysiert. Diese Promotoren waren, wie aus in vivo Expressionsstudien und DNA Schutzexperimenten bekannt, von unterschiedlicher Promotorstärke. Da die Initiation der Transkription aus verschiedenen nacheinander ablaufenden Schritten besteht (Promotorbindung der RNAP, Isomerisierung zum offenen Komplex und Bildung eines stabilen Elongationskomplexes) war bis dahin unklar, welches der geschwindigkeitsbestimmende Schritt der Gesamtreaktion ist. Für die Promotoren glnAp2 und nifL war die Promotorbindung der RNAP-sigma54 geschwindigkeitsbestimmend. Im Gegensatz dazu war für den nifH Promotor, der zwar eine starke Affinität zu RNAP-sigma54 besitzt, aber nur schwach in vivo exprimiert, das Aufschmelzen der DNA bestimmend für die Gesamtreaktion der Transkription. (2) Mittels Rasterkraftmikroskopie wurde festgestellt, dass die gebundene RNAP-sigma54 die DNA krümmt und deshalb vorzugsweise in der Endschleife einer superhelikalen DNA lokalisiert wird, da dort die DNA stärker gebogen ist. Diese Lokalisation in der Endschleife erleichtert die Interaktion zwischen NtrC and RNAP-sigma54 trotz der geringen Biegsamkeit der dazwischen liegenden DNA. (3) Die Interaktion zwischen sigma54 und NtrC wurde in einem ATPase-Test und in Gelshift-Experimenten analysiert. Es wurde festgestellt, dass sigma54 die ATPase-Aktivität von NtrC unter den Versuchsbedingungen nicht beeinflusst, während die Bindung von NtrC an den Enhancer die Aktivität durch Oligomerisierung von NtrC stark stimuliert. Bindungsstudien, die mit Hilfe der analytischen Ultrazentrifugation und Gelshift-Experimenten durchgeführt wurden, haben außerdem gezeigt, dass sigma54 allein den Promotor nur sehr schwach binden kann im Gegensatz zum RNAP-sigma54-Holoenzym. Dies unterstützt die Vorstellung, dass die Sigma-Untereinheit vor allem dazu dient, die Promotorsequenz zu erkennen, während das RNAP-Holoenzym die Bindungsenergie für eine hochaffine Promotorbindung liefert. (4) In in vitro Transkriptionsexperimenten wurde gezeigt, dass NtrC je nach Position, Anzahl und Zusammenstellung seiner Bindungsstellen die Transkription verschieden stark aktiviert oder sogar als Repressor wirken kann. Es zeigte sich, dass der Promotor mit bestimmten Kombinationen aus starken und schwachen NtrC-Bindungsstellen bei sehr unterschiedlichen NtrC-Konzentrationen aktiviert wird. Dies ermöglicht die Regulation der Transkription in Abhängigkeit von der NtrC-Konzentration. Darüber hinaus konnte gezeigt werden, dass eine Transkriptionaktivierung auch ohne DNA-Schleifenbildung bei höheren NtrC-Konzentrationen stattfindet. Anhand dieser Ergebnisse wurde ein Modell der RNAP-sigma54-NtrC-vermittelten Transkriptionsaktivierung erstellt: Demnach erleichtern NtrC-Bindungsstellen nahe des Promotors die Interaktion zwischen NtrC und RNA-Polymerase im Loopkomplex bei niedrigen NtrC-Konzentrationen, während sie bei höheren Konzentrationen die Transkriptionsaktivierung auf ein bestimmtes Maximum limitieren. In diesem Fall wird eine andere NtrC-Spezies gebildet, die ohne DNA-Schleifenbildung mit der RNAP-sigma54 interagieren kann.

Construction and functional analyses of a comprehensive 54 site-directed mutant library using alanine-cysteine mutagenesis

Article

Full-text available

Jun 2009
NUCLEIC ACIDS RES

The σ54 factor associates with core RNA polymerase (RNAP) to form a holoenzyme that is unable to initiate transcription unless acted on by an activator protein. σ54 is closely involved in many steps of activator-dependent transcription, such as core RNAP binding, promoter recognition, activator interaction and open complex formation. To systematically define σ54 residues that contribute to each of these functions and to generate a resource for site specific protein labeling, a complete mutant library of σ54 was constructed by alanine–cysteine scanning mutagenesis. Amino acid residues from 3 to 476 of Cys(-)σ54 were systematically mutated to alanine and cysteine in groups of two adjacent residues at a time. The influences of each substitution pair upon the functions of σ54 were analyzed in vivo and in vitro and the functions of many residues were revealed for the first time. Increased σ54 isomerization activity seldom corresponded with an increased transcription activity of the holoenzyme, suggesting the steps after σ54 isomerization, likely to be changes in core RNAP structure, are also strictly regulated or rate limiting to open complex formation. A linkage between core RNAP-binding activity and activator responsiveness indicates that the σ54-core RNAP interface changes upon activation.

Binding of transcriptional activators to sigma 54 in the presence of the transition state analog ADP-aluminum fluoride: Insights into activator mechanochemical action

Article

Full-text available

Oct 2001
GENE DEV

Conformational changes in sigma 54 (sigma(54)) and sigma(54)-holoenzyme depend on nucleotide hydrolysis by an activator. We now show that sigma(54) and its holoenzyme bind to the central ATP-hydrolyzing domains of the transcriptional activators PspF and NifA in the presence of ADP-aluminum fluoride, an analog of ATP in the transition state for hydrolysis. Direct binding of sigma(54) Region I to activator in the presence of ADP-aluminum fluoride was shown and inferred from in vivo suppression genetics. Energy transduction appears to occur through activator contacts to sigma(54) Region I. ADP-aluminum fluoride-dependent interactions and consideration of other AAA+ proteins provide insight into activator mechanochemical action.

Action of prokaryotic enhancer over a distance does not require continued presence of promoter-bound σ54 subunit

Article

Full-text available

Mar 2002
NUCLEIC ACIDS RES

The mechanism by which an enhancer activates transcription over large distances has been investigated. Activation of the glnAp2 promoter by the NtrC-dependent enhancer in Escherichia coli was analyzed using a purified system supporting multiple-round transcription in vitro. Our results suggest that the enhancer–promoter interaction and the initiation complex must be formed de novo during every round of transcription. No protein remained bound to the promoter after RNA polymerase escaped into elongation. Furthermore, the rate of initiation during the first and subsequent rounds of transcription were very similar, suggesting that there was no functional ‘memory’ facilitating multiple rounds of transcription. These studies exclude the hypothesis that enhancer action during multiple-round transcription involves the memory of the initial activation event.

Correlating protein footprinting with mutational analysis in the bacterial transcription factor σ54 (σN)

Article

Full-text available

Mar 2002
NUCLEIC ACIDS RES

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.

β Subunit Residues 186–433 and 436–445 are Commonly Used by Eσ54 and Eσ70 RNA Polymerase for Open Promoter Complex Formation

Article

Jul 2002

During transcription initiation by DNA-dependent RNA polymerase (RNAP) promoter DNA has to be melted locally to allow the synthesis of RNA transcript. Localized melting of promoter DNA is a target for genetic regulation and is poorly understood at the molecular level. The Escherichia coli RNAP holoenzyme is a six-subunit (alpha(2)betabeta'omegasigma; Esigma) protein complex. The sigma subunit is directly responsible for promoter recognition and contributes to localized DNA melting. Mutations in the beta subunit have profound effects on promoter melting by Esigma70. The sigma54 subunit is a representative of an unrelated class of the sigma subunits. Here, we determined whether mutations in the beta subunit that affect late stages of promoter complex formation by Esigma70 also influence promoter complex formation by the enhancer-dependent Esigma54. Analyses of in vitro defects in promoter complex formation and transcription initiation exhibited by mutant Esigma54 suggest that during promoter complex formation by Esigma54 and Esigma70 a common set of beta subunit sequences is used. Late stages of promoter complex formation and localized melting of promoter DNA by Esigma70 and Esigma54 thus proceed through a common pathway.

Transcriptional regulation of soluble methane monooxygenase via enhancer-binding protein derived from Methylosinus sporium 5

Article

Full-text available

Sep 2023
APPL ENVIRON MICROB

Methane is a major greenhouse gas, and methanotrophs regulate the methane level in the carbon cycle. Soluble methane monooxygenase (sMMO) is expressed in various methanotroph genera, including Alphaproteobacteria and Gammaproteobacteria , and catalyzes the hydroxylation of methane to methanol. It has been proposed that MmoR regulates the expression of sMMO as an enhancer-binding protein under copper-limited conditions; however, details on this transcriptional regulation remain limited. Herein, we elucidate the transcriptional pathway of sMMO depending on copper ion concentration, which affects the interaction of MmoR and sigma factor. MmoR and sigma-54 (σ ⁵⁴ ) from Methylosinus sporium 5 were successfully overexpressed in Escherichia coli and purified to investigate sMMO transcription in methanotrophs. The results indicated that σ ⁵⁴ binds to a promoter positioned −24 (GG) and −12 (TGC) upstream between mmoG and mmoX1 . The binding affinity and selectivity are lower ( K d = 184.6 ± 6.2 nM) than those of MmoR. MmoR interacts with the upstream activator sequence (UAS) with a strong binding affinity ( K d = 12.5 ± 0.5 nM). Mutational studies demonstrated that MmoR has high selectivity to its binding partner (ACA-xx-TGT). Titration assays have demonstrated that MmoR does not coordinate with copper ions directly; however, its binding affinity to UAS decreases in a low-copper-containing medium. MmoR strongly interacts with adenosine triphosphate ( K d = 62.8 ± 0.5 nM) to generate RNA polymerase complex. This study demonstrated that the binding events of both MmoR and σ ⁵⁴ that regulate transcription in M. sporium 5 depend on the copper ion concentration. IMPORTANCE This study provides biochemical evidence of transcriptional regulation of soluble methane monooxygenase (sMMO) in methanotrophs that control methane levels in ecological systems. Previous studies have proposed transcriptional regulation of MMOs, including sMMO and pMMO, while we provide further evidence to elucidate its mechanism using a purified enhancer-binding protein (MmoR) and transcription factor (σ ⁵⁴ ). The characterization studies of σ ⁵⁴ and MmoR identified the promoter binding sites and enhancer-binding sequences essential for sMMO expression. Our findings also demonstrate that MmoR functions as a trigger for sMMO expression due to the high specificity and selectivity for enhancer-binding sequences. The UV-visible spectrum of purified MmoR suggested an iron coordination like other GAF domain, and that ATP is essential for the initiation of enhancer elements. Binding assays indicated that these interactions are blocked by the copper ion. These results provide novel insights into gene regulation of methanotrophs.

Document S2. Article plus Supplemental Information

Data

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Jul 2017

Structures of RNA Polymerase Closed and Intermediate Complexes Reveal Mechanisms of DNA Opening and Transcription Initiation

Article

Full-text available

Jun 2017
MOL CELL

Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ⁵⁴ factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ⁵⁴ interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ⁵⁴ that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo.

The bacterial enhancer-dependent RNA polymerase

Article

Full-text available

Nov 2016

Transcription initiation is highly regulated in bacterial cells, allowing adaptive gene regulation in response to environment cues. One class of promoter specificity factor called sigma54 enables such adaptive gene expression through its ability to lock the RNA polymerase down into a state unable to melt out promoter DNA for transcription initiation. Promoter DNA opening then occurs through the action of specialized transcription control proteins called bacterial enhancer-binding proteins (bEBPs) that remodel the sigma54 factor within the closed promoter complexes. The remodelling of sigma54 occurs through an ATP-binding and hydrolysis reaction carried out by the bEBPs. The regulation of bEBP self-assembly into typically homomeric hexamers allows regulated gene expression since the self-assembly is required for bEBP ATPase activity and its direct engagement with the sigma54 factor during the remodelling reaction. Crystallographic studies have now established that in the closed promoter complex, the sigma54 factor occupies the bacterial RNA polymerase in ways that will physically impede promoter DNA opening and the loading of melted out promoter DNA into the DNA-binding clefts of the RNA polymerase. Large-scale structural re-organizations of sigma54 require contact of the bEBP with an amino-terminal glutamine and leucine-rich sequence of sigma54, and lead to domain movements within the core RNA polymerase necessary for making open promoter complexes and synthesizing the nascent RNA transcript.

Genome wide interactions of wild-type and activator bypass forms of 54

Article

Full-text available

Jun 2015
NUCLEIC ACIDS RES

Enhancer-dependent transcription involving the promoter specificity factor σ(54) is widely distributed amongst bacteria and commonly associated with cell envelope function. For transcription initiation, σ(54)-RNA polymerase yields open promoter complexes through its remodelling by cognate AAA+ ATPase activators. Since activators can be bypassed in vitro, bypass transcription in vivo could be a source of emergent gene expression along evolutionary pathways yielding new control networks and transcription patterns. At a single test promoter in vivo bypass transcription was not observed. We now use genome-wide transcription profiling, genome-wide mutagenesis and gene over-expression strategies in Escherichia coli, to (i) scope the range of bypass transcription in vivo and (ii) identify genes which might alter bypass transcription in vivo. We find little evidence for pervasive bypass transcription in vivo with only a small subset of σ(54) promoters functioning without activators. Results also suggest no one gene limits bypass transcription in vivo, arguing bypass transcription is strongly kept in check. Promoter sequences subject to repression by σ(54) were evident, indicating loss of rpoN (encoding σ(54)) rather than creating rpoN bypass alleles would be one evolutionary route for new gene expression patterns. Finally, cold-shock promoters showed unusual σ(54)-dependence in vivo not readily correlated with conventional σ(54) binding-sites. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Multiple Roles of the RNA Polymerase β Subunit Flap Domain in ς54-Dependent Transcription

Article

Jan 2003

Recent determinations of the structures of the bacterial RNA polymerase (RNAP) and promoter complex thereof establish that RNAP functions as a complex molecular machine that contains distinct structural modules that undergo major conformational changes during transcription. However, the contribution of the RNAP structural modules to transcription remains poorly understood. The bacterial core RNAP (α2ββ′ω; E) associates with a sigma (ς) subunit to form the holoenzyme (Eς). A mutation removing the β subunit flap domain renders the Escherichia coliς70 RNAP holoenzyme unable to recognize promoters. ς54 is the major variant ς subunit that utilizes enhancer-dependent promoters. Here, we determined the effects of β flap removal on ς54-dependent transcription. Our analysis shows that the role of the β flap in ς54-dependent and ς70-dependent transcription is different. Removal of the β flap does not prevent the recognition of ς54-dependent promoters, but causes multiple defects in ς54-dependent transcription. Most importantly, the β flap appears to orchestrate the proper formation of the Eς54 regulatory center at the start site proximal promoter element where activator binds and DNA melting originates.

A Gal4-σ54 Hybrid Protein that Functions as a Potent Activator of RNA Polymerase II Transcription in Yeast

Article

Full-text available

Jul 2001

The bacterial ς54 protein associates with core RNA polymerase to form a holoenzyme complex that renders cognate promoters enhancer-dependent. Although unusual in bacteria, enhancer-dependent transcription is the paradigm in eukaryotes. Here we report that a fragment ofEscherichia coli ς54 encompassing amino acid residues 29–177 functions as a potent transcriptional activator in yeast when fused to a Gal4 DNA binding domain. Activation by Gal4-ς54 is TATA-dependent and requires the SAGA coactivator complex, suggesting that Gal4-ς54functions by a normal mechanism of transcriptional activation. Surprisingly, deletion of the AHC1 gene, which encodes a polypeptide unique to the ADA coactivator complex, stimulates Gal4-ς54-mediated activation and enhances the toxicity of Gal4-ς54. Accordingly, the SAGA and ADA complexes, both of which include Gcn5 as their histone acetyltransferase subunit, exert opposite effects on transcriptional activation by Gal4-ς54. Gal4-ς54 activation and toxicity are also dependent upon specific ς54 residues that are required for activator-responsive promoter melting by ς54in bacteria, implying that activation is a consequence of ς54-specific features rather than a structurally fortuitous polypeptide fragment. As such, Gal4-ς54represents a novel tool with the potential to provide insight into the mechanism by which natural activators function in eukaryotic cells.

New Roles for Conserved Regions within a 54-dependent Enhancer-binding Protein

Article

Full-text available

Dec 2002

23 amino acid substitutions were made in the C7 and C3 regions of pspFDeltaHTH, a protein required to convert sigma(54) closed promoter complexes to open complexes. These mutants were assayed for transcriptional competence, for the ability to hydrolyze ATP, for their multimerization state, and for their ability to interact with sigma(54) and its holoenzyme. C7 region mutants caused the protein to assume a compact form. This property could be mimicked by the addition of ATP, implying that compaction via C7 and ATP is part of the activation process. A number of C3 mutants were important for energy coupling, as indicated previously for several members of this activator family (, ). However, a patch within C3 influenced oligomerization. The C3 region was especially important in interacting with sigma(54) during the transition state but not important in inducing sigma(54) holoenzyme to engage the nontemplate strand of the promoter. It is proposed that both regions contain deterrent functions that prevent premature activation. Overall, the results imply unexpected roles for the C7 and C3 regions of this protein family during promoter activation.

Multiple Roles of the RNA Polymerase Subunit Flap Domain in 54-Dependent Transcription

Article

Full-text available

Feb 2003

Recent determinations of the structures of the bacterial RNA polymerase (RNAP) and promoter complex thereof establish that RNAP functions as a complex molecular machine that contains distinct structural modules that undergo major conformational changes during transcription. However, the contribution of the RNAP structural modules to transcription remains poorly understood. The bacterial core RNAP (alpha(2)beta beta'omega; E) associates with a sigma (sigma) subunit to form the holoenzyme (E sigma). A mutation removing the beta subunit flap domain renders the Escherichia coli sigma(70) RNAP holoenzyme unable to recognize promoters. sigma(54) is the major variant sigma subunit that utilizes enhancer-dependent promoters. Here, we determined the effects of beta flap removal on sigma(54)-dependent transcription. Our analysis shows that the role of the beta flap in sigma(54)-dependent and sigma(70)-dependent transcription is different. Removal of the beta flap does not prevent the recognition of sigma(54)-dependent promoters, but causes multiple defects in sigma(54)-dependent transcription. Most importantly, the beta flap appears to orchestrate the proper formation of the E sigma(54) regulatory center at the start site proximal promoter element where activator binds and DNA melting originates.

Regulation of the transcriptional activator NtrC1: Structural studies of the regulatory and AAA+ ATPase domains

Article

Full-text available

Nov 2003
GENE DEV

Transcription by sigma54 RNA polymerase depends on activators that contain ATPase domains of the AAA+ class. These activators, which are often response regulators of two-component signal transduction systems, remodel the polymerase so that it can form open complexes at promoters. Here, we report the first crystal structures of the ATPase domain of an activator, the NtrC1 protein from the extreme thermophile Aquifex aeolicus. This domain alone, which is active, crystallized as a ring-shaped heptamer. The protein carrying both the ATPase and adjacent receiver domains, which is inactive, crystallized as a dimer. In the inactive dimer, one residue needed for catalysis is far from the active site, and extensive contacts among the domains prevent oligomerization of the ATPase domain. Oligomerization, which completes the active site, depends on surfaces that are buried in the dimer, and hence, on a rearrangement of the receiver domains upon phosphorylation. A motif in the ATPase domain known to be critical for coupling energy to remodeling of polymerase forms a novel loop that projects from the middle of an alpha helix. The extended, structured loops from the subunits of the heptamer localize to a pore in the center of the ring and form a surface that could contact sigma54.

The Second Paradigm for Activation of Transcription

Article

Feb 2005
PROG MOL BIOL TRANSL

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.

Interactions of regulated and deregulated forms of the σ54 holoenzyme with heteroduplex promoter DNA

Article

Full-text available

The bacterial σ54 RNA polymerase holoenzyme binds to promoters as a stable closed complex that is silent for transcription unless acted upon by an enhancer- bound activator protein. Using DNA binding and tran- scription assays the ability of the enhancer- dependent σ54 holoenzyme to interact with promoter DNA containing various regions of heteroduplex from -12 to -1 was assessed. Different DNA regions important for stabilising σ54 holoenzyme-promoter interactions, destabilising binding, limiting template utilisation in activator-dependent transcription and for stable binding of a deregulated form of the holoenzyme lacking σ54 Region I were identified. It appears that homoduplex structures are required for early events in σ54 holoenzyme promoter binding and that disruption of a repressive fork junction structure only modestly deregulates transcription. DNA opening from -5 to -1 appears important for stable engagement of the holoenzyme following activation. The regulatory Region I of σ54 was shown to be involved in interactions with the sequences in the -5 to -1 area.

Escherichia coli promoter opening and -10 recognition: Mutational analysis of ??70

Article

Mar 2000
EMBO J

The opening of specific segments of DNA is required for most types of genetic readout, including 70-dependent transcription. To learn how this occurs, a series of single point mutations were introduced into 70 region 2. These were assayed for duplex DNA binding, DNA opening and DNA double strand–single strand fork junction binding. Band shift assays for closed complex formation implicated a series of arginine and aromatic residues within a minimal 26 amino acid region. Permanganate assays implicated two additional aromatic residues in DNA opening, known to form a parallel stack of the type that can accept a flipped-out base. Substitution for either of these aromatics had no effect on duplex probe recognition. However, when a single unpaired -11 nucleotide is added to the probe, the mutants fail to bind appropriately to give heparin resistance. A model for DNA opening is presented in which duplex recognition by regions 2.3, 2.4 and 2.5 of sigma positions the pair of aromatic amino acids, which then create the fork junction required for stable opening.

The Role of Bacterial Enhancer Binding Proteins as Specialized Activators of 54-Dependent Transcription

Article

Full-text available

Sep 2012

Bacterial enhancer binding proteins (bEBPs) are transcriptional activators that assemble as hexameric rings in their active forms and utilize ATP hydrolysis to remodel the conformation of RNA polymerase containing the alternative sigma factor σ54. We present a comprehensive and detailed summary of recent advances in our understanding of how these specialized molecular machines function. The review is structured by introducing each of the three domains in turn: the central catalytic domain, the N-terminal regulatory domain, and the C-terminal DNA binding domain. The role of the central catalytic domain is presented with particular reference to (i) oligomerization, (ii) ATP hydrolysis, and (iii) the key GAFTGA motif that contacts σ54 for remodeling. Each of these functions forms a potential target of the signal-sensing N-terminal regulatory domain, which can act either positively or negatively to control the activation of σ54-dependent transcription. Finally, we focus on the DNA binding function of the C-terminal domain and the enhancer sites to which it binds. Particular attention is paid to the importance of σ54 to the bacterial cell and its unique role in regulating transcription.

Análise estrutural e funcional do gene rpoN de Herbaspirillum seropedicae /

Thesis

Jan 2006

Fabiane Gomes de Moraes Rego

Orientador : Emanuel M. de Souza Co-orientador : Fabio O. Pedrosa Tese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica. Defesa: Curitiba, 2006 Inclui bibliografia

Regulation of Sigma 54-Dependent Transcription by Core Promoter Sequences: Role of −12 Region Nucleotides

Article

Jan 2000

The tetranucleotide core recognition sequence (TTGC) of the sigma 54 promoter -12 recognition element was altered by random substitution. The resulting promoter mutants were characterized in vivo and in vitro. Deregulated promoters were identified, implying that this core element can mediate the response to enhancer-binding proteins. These promoters had in common a substitution at position -12 (consensus C), indicating its importance for keeping basal transcription in check. In another screen, nonfunctional promoters were identified. Their analysis indicated that positions -13 (consensus G) and -15 (consensus T) are important to maintain minimal promoter function. In vitro studies showed that the -13 and -15 positions contribute to closed-complex formation, whereas the -12 position has a stronger effect on recognition of the fork junction intermediate created during open-complex formation. Overall the data indicate that the -12 region core contains specific subsequences that direct the diverse RNA polymerase interactions required both to produce RNA and to restrict this RNA synthesis in the absence of activation.

Functionality of purified sigma(N) (sigma(54)) and a NifA-like protein from the hyperthermophile Aquifex aeolicus

Article

Full-text available

Mar 2000
J BACTERIOL

The genome sequence of the extremely thermophilic bacteriumAquifex aeolicus encodes alternative sigma factor ςN (ς54, RpoN) and five potential ςN-dependent transcriptional activators. AlthoughA. aeolicus possesses no recognizable nitrogenase genes, two of the activators have a high degree of sequence similarity to NifA proteins from nitrogen-fixing proteobacteria. We identified five putative ςN-dependent promoters upstream of operons implicated in functions including sulfur respiration, nitrogen assimilation, nitrate reductase, and nitrite reductase activity. We cloned, overexpressed (in Escherichia coli), and purifiedA. aeolicus ςN and the NifA homologue, AQ_218. Purified A. aeolicus ςN bound toE. coli core RNA polymerase and bound specifically to a DNA fragment containing E. coli promoter glnHp2 and to several A. aeolicus DNA fragments containing putative ςN-dependent promoters. When combined with E. coli core RNA polymerase, A. aeolicusςN supported A. aeolicus NifA-dependent transcription from the glnHp2 promoter. The E. coli activator PspFΔHTH did not stimulate transcription. The NifA homologue, AQ_218, bound specifically to a DNA sequence centered about 100 bp upstream of the A. aeolicus glnBA operon and so is likely to be involved in the regulation of nitrogen assimilation in this organism. These results argue that the ςNenhancer-dependent transcription system operates in at least one extreme environment, and that the activator and ςN have coevolved.

Sequences in σ54 region I required for binding to early melted DNA and their involvement in sigma-DNA isomerisation

Article

May 2000

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.

Sequences within the DNA Cross-linking Patch of ς 54 Involved in Promoter Recognition, ς Isomerization, and Open Complex Formation

Article

Full-text available

Aug 2000

The bacterial RNA polymerase holoenzyme containing the ς54 subunit functions in enhancer-dependent transcription. Mutagenesis has been used to probe the function of a sequence in the ς54 DNA binding domain that includes residues that cross-link to promoter DNA. Several activities of the ς and holoenzyme are shown to depend on the cross-linking patch. The patch contributes to promoter binding by ς54, and holoenzyme and is involved in activator-dependent ς isomerization. As part of the ς54-holoenzyme, some residues in the patch limit basal transcription. Other cross-linking patch sequences appear to limit activator-dependent open complex formation. Deletion of 19 residues adjacent to the cross-linking patch resulted in a holoenzyme unable to respond to activator but capable of activator-independent (bypass) transcription in vitro. Overall results are consistent with the cross-linking patch directing interactions to the −12 promoter region to set basal and activated levels of transcription.

Signaling through sigma

Article

Aug 2000
Nat Struct Biol

Jay D. Gralla

Some prokaryotic transcriptional activators act by binding to enhancers and directly changing the conformation of a specialized sigma factor in the RNA polymerase holoenzyme. This mechanism has interesting parallels in other transcription systems.

The Bacterial Enhancer-Dependent ς54(ςN) Transcription Factor

Article

Full-text available

Aug 2000
J BACTERIOL

DNA melting within a binary σ54-promoter DNA complex

Article

Full-text available

Feb 2001

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.

Single amino acid substitution mutants of Klebsiella pneumoniae σ54 defective in transcription

Article

Full-text available

Dec 2000
NUCLEIC ACIDS RES

Transcription initiation by the sigma(54) RNA polymerase requires specialised activators and their associated nucleoside triphosphate hydrolysis. To explore the roles of sigma(54) in initiation we used random mutagenesis of rpoN and an in vivo activity screen to isolate functionally altered sigma(54) proteins. Five defective mutants, each with a different single amino acid substitution, were obtained. Three failed in transcription after forming a closed complex. One such mutant mapped to regulatory Region I of sigma(54), the other two to Region III. The Region I mutant allowed transcription independently of activator and showed reduced activator-dependent sigma(54) isomerisation. The two Region III mutants displayed altered behaviour in a sigma(54) isomerisation assay and one failed to stably bind early melted DNA as the holoenzyme; they may contribute to a communication pathway linking changes in sigma to open complex formation. Two further Region III mutants showed gross defects in overall DNA binding. For one, sufficient residual DNA binding activity remained to allow us to demonstrate that other activities were largely unaffected. Changes in DNA binding preferences and core polymerase-dependent properties were evident amongst the mutants.

Regulatory sequences in sigma 54 localise near the start of DNA melting

Article

Apr 2001

Transcription initiation by the enhancer-dependent sigma(54) RNA polymerase holoenzyme is positively regulated after promoter binding. The promoter DNA melting process is subject to activation by an enhancer-bound activator protein with nucleoside triphosphate hydrolysis activity. Tethered iron chelate probes attached to amino and carboxyl-terminal domains of sigma(54) were used to map sigma(54)-DNA interaction sites. The two domains localise to form a centre over the -12 promoter region. The use of deletion mutants of sigma(54) suggests that amino-terminal and carboxyl-terminal sequences are both needed for the centre to function. Upon activation, the relationship between the centre and promoter DNA changes. We suggest that the activator re-organises the centre to favour stable open complex formation through adjustments in sigma(54)-DNA contact and sigma(54) conformation. The centre is close to the active site of the RNA polymerase and includes sigma(54) regulatory sequences needed for DNA melting upon activation. This contrasts systems where activators recruit RNA polymerase to promoter DNA, and the protein and DNA determinants required for activation localise away from promoter sequences closely associated with the start of DNA melting.

Control of nuclear export of hnRNP A1

Article

May 2001

mRNA export is mediated by RNA-binding proteins which shuttle between the nucleus and cytoplasm. Using an in vitro unidirectional export assay, we observe that the shuttling mRNA-binding protein, hnRNP A1, is exported only extremely slowly unless incubations are supplemented with snRNA-specific oligonucleotides which inhibit splicing. In vivo microinjection experiments support this conclusion. Like many examples of nucleocytoplasmic transport, export of hnRNP A1 requires energy and is sensitive to the presence of wheat germ agglutinin. It does not, however, require supplementation with cytoplasmic proteins. Although the exportin, Crm1, is needed for export of several varieties of RNA, both the in vitro assay and in vivo assays show that it is not required for export of hnRNP A1. In vitro and in vivo studies also show that inhibition of transcription allows continued shuttling of hnRNP A1 and in fact accelerates its export. Judging from the stimulatory effects of targeted destruction of snRNAs, this is likely to reflect completion of the covalent maturation of the RNAs with which hnRNP A1 associates. These observations therefore provide a simple explanation of why multiple RNA-binding proteins relocate to the cytoplasm upon inhibition of transcription in vivo.

Sigma38 (rpoS) RNA Polymerase Promoter Engagement via - 10 Region Nucleotides

Article

Full-text available

Sep 2001

Band shift assays using DNA probes that mimic closed and open complexes were used to explore the determinants of promoter recognition by sigma38 (rpoS) RNA polymerase. Duplex recognition was found to be much weaker than that observed in sigma70 promoter usage. However, binding to fork junction probes, which attempt to mimic melted DNA, was very strong. This binding occurs via the non-template strand with the identity of the two conserved junction nucleotides (−12T and −11A) being of paramount importance. A modified promoter consensus sequence identified these two nucleotides as among only four (underlined) that are highly conserved, and all four were in the −10 region (CTAcacT from −13 to −7). The remaining two nucleotides were shown to have different roles, −13C in preventing recognition by the heterologous sigma70 polymerase and −7T in directing enzyme isomerization. These −10 region nucleotides appear to have their primary function prior to full melting because probes that had a melted start site were relatively insensitive to substitution at these positions. These results suggest the sigma38 mechanism differs from the sigma70 mechanism, and this difference likely contributes to selective use of sigma38 under conditions that exist during stationery phase.

DNA supercoiling allows enhancer action over a large distance

Article

Full-text available

Jan 2002

Enhancers are regulatory DNA elements that can activate their genomic targets over a large distance. The mechanism of enhancer action over large distance is unknown. Activation of the glnAp2 promoter by NtrC-dependent enhancer in Escherichia coli was analyzed by using a purified system supporting multiple-round transcription in vitro. The data suggest that DNA supercoiling is an essential requirement for enhancer action over a large distance (2,500 bp) but not over a short distance (110 bp). DNA supercoiling facilitates functional enhancer-promoter communication over a large distance, probably by bringing the enhancer and promoter into close proximity.

The regulatory N-terminal region of the aromatic-responsive transcriptional activator DmpR constrains nucleotide-triggered multimerisation

Article

Jan 2002

The transcriptional promoting activity of DmpR is under the strict control of its aromatic effector ligands that are bound by its regulatory N-terminal domain. The positive control function of DmpR resides within the central C-domain that is highly conserved among activators of sigma(54)-RNA polymerase. The C-domain mediates ATP hydrolysis and interaction with sigma(54)-RNA polymerase that are essential for open-complex formation and thus initiation of transcription. Wild-type and loss-of-function derivatives of DmpR, which are defective in distinct steps in nucleotide catalysis, were used to address the consequences of nucleotide binding and hydrolysis with respect to the multimeric state of DmpR and its ability to promote in vitro transcription. Here, we show that DmpR derivatives deleted of the regulatory N-terminal domain undergo an aromatic-effector independent ATP-binding triggered multimerisation as detected by cross-linking. In the intact protein, however, aromatic effector activation is required before ATP-binding can trigger an apparent dimer-to-hexamer switch in subunit conformation. The data suggest a model in which the N-terminal domain controls the transcriptional promoting property of DmpR by constraining ATP-mediated changes in its oligomeric state. The results are discussed in the light of recent mechanistic insights from the AAA(+) superfamily of ATPases that utilise nucleotide hydrolysis to restructure their substrates.

The Energetics of Consensus Promoter Opening by T7 RNA Polymerase

Article

Dec 2002

The consensus 23 base-pair T7 DNA promoter is classically divided into two domains, an upstream binding domain (-17 to -5), and a downstream initiation domain (-4 to +6) relative to the transcription start site at +1. During transcription initiation, T7 RNA polymerase (T7 RNAP) melts specifically the -4 to +2/+3 (TATAGG/G) region of the duplex DNA promoter to form a pre-initiation open complex. No external energy source is used and the energy for open complex formation is derived from the free energy of specific interactions with the binding domain, particularly the specificity region (-13 to -6). Using 2-aminopurine fluorescence-based equilibrium and kinetic measurements, we have measured the binding affinities of various topologically modified DNA promoters (40 bp in length) that represent initial, final, and transition-state analogs of the promoter DNA in the T7 RNAP-DNA complex, to determine the energy of specific binding interactions, and the energy required for forming an initiation bubble. The results indicate that 16-16.5 kcal mol(-1) of free energy is made available upon T7 RNAP binding (through specificity loop) to the promoter binding domain. To melt the TATAGG/G sequence 7-8 kcal mol(-1) of free energy is utilized; this compares with approximately 6 kcal mol(-1) predicted from nearest neighbor analysis. The remaining 8.5-9.5 kcal mol(-1) of net free energy is retained for stabilization of the specific pre-initiation binary complex. Of the 7-8 kcal mol(-1) energy that is used to generate the pre-initiation DNA bubble in the open complex, we estimate that one half (3.5-4 kcal mol(-1)) is utilized for nucleation/deformation process (through bending, untwisting, etc.) in the melting region (-4 to -1 TATA) of the initiation domain (-4 to +6), and appears to be independent of the nucleation site within this region. The other half is utilized in unpairing the +1 to +2/+3 GG/G sequence for initiation. The interactions of T7 RNAP with a 20-bp non-specific DNA on the other hand are very weak (DeltaG<-5k cal mol(-1)), which is not sufficient to melt and stabilize an open complex of a non-specific DNA.

Mapping 54-RNA Polymerase Interactions at the -24 Consensus Promoter Element

Article

Full-text available

Sep 2003

The sigma 54 promoter specificity factor is distinct from sigma 70-type factors. The sigma 54-RNA polymerase binds to promoters with conserved sequence elements at -24 and -12 and utilizes specialized enhancer-binding activators to convert, through an ATP-dependent process, closed promoter complexes to open promoter complexes. The interface between sigma 54-RNA polymerase and promoter DNA is poorly characterized, contrasting with sigma 70. Here, sigma 54 was modified with strategically positioned cleavage reagents to provide physical evidence that the highly conserved RpoN box motif of sigma 54 is close to and may therefore interact with the consensus -24 promoter element. We show that the spatial relationship between the sigma 54-RNA polymerase and the -24 promoter element remains unchanged during closed to open complex conversion and transcription initiation but changes during the early elongation phase. In contrast, the spatial relationship between sigma 54-RNA polymerase and the consensus -12 promoter element changes upon conversion of the closed promoter complex to an open one. We provide evidence that some -12 promoter region-sigma 54 interactions are dependent upon either the core RNA polymerase or a fork junction DNA structure at the -12-position, indicating that DNA fork junctions can substitute for core RNAP. We also show the beta-subunit flap domain contributes to different sets of sigma-promoter DNA interactions at sigma 54- and sigma 70-dependent promoters.

Effect of DNA bases and backbone on σ70 holoenzyme binding and isomerization using fork junction probes

Article

Jul 2003
NUCLEIC ACIDS RES

Abasic substitutions in the non‐template strand and promoter sequence changes were made to assess the roles of various promoter features in σ70 holoenzyme interactions with fork junction probes. Removal of –10 element non‐template single strand bases, leaving the phosphodiester backbone intact, did not interfere with binding. In contrast these abasic probes were deficient in promoting holoenzyme isomerization to the heparin resistant conformation. Thus, it appears that the melted –10 region interaction has two components, an initial enzyme binding primarily to the phosphodiester backbone and a base dependent isomerization of the bound enzyme. In contrast various upstream elements cooperate primarily to stimulate binding. Features and positions most important for these effects are identified.

Communication over a large distance: Enhancers and insulators

Article

Jul 2003

Enhancers are regulatory DNA sequences that can work over a large distance. Efficient enhancer action over a distance clearly requires special mechanisms for facilitating communication between the enhancer and its target. While the chromatin looping model can explain the majority of the observations, some recent experimental findings suggest that a chromatin scanning mechanism is used to establish the loop. These new findings help to understand the mechanism of action of the elements that can prevent enhancer-promoter communication (insulators).

A protein-induced DNA bend increases the specificity of a prokaryotic enhancer-binding protein

Article

Full-text available

Mar 1998
GENE DEV

Control of transcription in prokaryotes often involves direct contact of regulatory proteins with RNA polymerase from binding sites located adjacent to the target promoter. Alternatively, in the case of genes transcribed by Escherichia coli RNA polymerase holoenzyme containing the alternate sigma factor sigma54, regulatory proteins bound at more distally located enhancer sites can activate transcription via DNA looping by taking advantage of the increasing flexibility of DNA over longer distances. While this second mechanism offers a greater possible flexibility in the location of these binding sites, it is not clear how the specificity offered by the proximity of the regulatory protein and the polymerase intrinsic to the first mechanism is maintained. Here we demonstrate that integration host factor (IHF), a protein that induces a sharp bend in DNA, acts both to inhibit DNA-looping-dependent transcriptional activation by an inappropriate enhancer-binding protein and to facilitate similar activation by an appropriate enhancer-binding protein. These opposite effects have the consequence of increasing the specificity of activation of a promoter that is susceptible to regulation by proteins bound to a distal site.

The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent

Article

Full-text available

Jul 1992

The prokaryotic activator protein NTRC binds to enhancer-like elements and activates transcription in response to nitrogen limitation by catalysing open complex formation by sigma 54 RNA polymerase holoenzyme. Formation of open complexes requires the phosphorylated form of NTRC and the reaction is ATP dependent. We find that NTRC has an ATPase activity which is activated by phosphorylation and is strongly stimulated by the presence of DNA containing specific NTRC binding sites.

Covalent Modification of the glnG Product, NRI, by the glnL Product, NRII, Regulates the Transcription of the glnALG Operon in Escherichia coli

Article

Full-text available

Sep 1986

Transcription from nitrogen-regulated promoters, such as glnAp2, requires the glnG gene product, NRI, as well as the rpoN(glnF) gene product, sigma60, and is regulated by the glnL gene product, NRII. We find that in a reaction mixture containing NRI, NRII, and ATP, NRII catalyzes the transfer of the gamma phosphate of ATP to NRI. This covalent modification of NRI occurs concurrently with the acquisition of the ability by the reaction mixture to activate transcription from glnAp2. In the presence of PII, the product of glnB, NRII catalyzes the removal of the phosphate from NRI-phosphate. This reaction occurs concurrently with the loss by the reaction mixture of the ability to activate transcription from glnAp2. On the basis of this evidence, we propose that NRI-phosphate activates transcription from nitrogen-regulated promoters and that the role of NRII is control of the formation and breakdown of NRI-phosphate in response to cellular signals of nitrogen availability.

Probing the Escherichia coli glnALG Upstream Activation Mechanism in vivo

Article

Full-text available

Jan 1989

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 vitro transcription close to the melting point of DNA: Analysis of Thermotoga maritima RNA polymerase-promoter complexes at 75°C using chemical probes

Article

Full-text available

Apr 1995

ABSTRACT The interaction of DNA dependent RNA polymerase of the extreme thermophile bacteria Thermotoga maritima with a promoter bearing DNA fragment was investigated in the temperature range from 20 to 85°C. We show that the T.maritima RNA polymerase recognizes and utilizes the Escherichia coli T7 A1 promoter with an efficiency similar to that of the E.coli polymerase. We have investigated the interaction of both polymerases with the same promoter over a wide range of temperatures using hydroxyl radical foot-printing and osmium tetroxide probing. This study revealed that the T.maritima polymerase goes through a series of isomerisation events very similarto the E.coli polymerase, i.e. the closed, intermediate and open complexes, but the transitions themselves occur at radically different temperatures. This indicates that conformational changes in the DNA that accompany initiation of transcription such as promoter melting are determined by the polymerase rather than the DNA sequence.

DNA distortion and nucleation of local DNA unwinding within Sigma-54 (σN) holoenzyme closed promoter complexes

Article

Full-text available

May 1994

The sigma N (sigma 54) RNA polymerase holoenzyme has the distinctive property of binding to promoters to form a closed promoter complex, which only isomerizes to the open complex when acted upon by an enhancer binding activator protein. We probed promoter complexes that form between sigma N and its holoenzyme with the conformationally sensitive footprinting reagents ortho-copper phenanthroline, potassium permanganate, and diethylpyrocarbonate. Results from these experiments indicate that the contacts sigma N makes at the -12 promoter element are necessary to promote a local DNA distortion immediately adjacent to this promoter element when the holoenzyme but not sigma N alone binds promoter DNA. Complexes in which this local distortion is not detected are not activatable, and the altered DNA conformation is diminished in the activated complex. We propose that a barrier to open complex formation in the sigma N holoenzyme closed complex is at some step or steps after the initial nucleation of DNA strand separation, which is detected as an altered DNA conformation stably maintained within the closed complex. Thus the activator protein may promote a conformational change in the sigma N holoenzyme to allow propagation of the altered DNA conformation, probably local unwinding, which we propose is necessary for formation of the melted DNA state, characteristic of the open promoter complex.

Base-Specific Recognition of the Nontemplate Strand of Promoter DNA by E. coli RNA Polymerase

Article

Full-text available

Sep 1996
CELL

RNA polymerase recognizes its promoters through base-specific interaction between defined segments of DNA and the sigma subunit of the enzyme. This interaction leads to separation of base pairs and exposure of the template strand for RNA synthesis. We show that base-specific recognition by the sigma 70 holoenzyme in this process involves primarily nontemplate strand bases in the -10 promoter region. We suggest that melting involves the persistence of these contacts as the bound duplex (closed) form is converted to the single-stranded (open) form of the enzyme-promoter complex.

The General transcription factors of RNA polymerase II

Article

Full-text available

Dec 1996
GENE DEV

Nucleoprotein complex formation by the enhancer binding protein NIFA

Article

Full-text available

Oct 1997

The nitrogen fixation protein NifA is a member of the protein family activating transcription by the alternative eubacterial sigmaN (sigma54) RNA polymerase holoenzyme. Binding sites for NifA, upstream activator sequences (UASs), are remotely located. Interaction between holoenzyme bound in a closed promoter complex and NiFA is facilitated by bending of the intervening DNA by integration host factor (IHF). We have examined NifA contact with the Klebsiella pneumoniae nifH promoter UAS in the presence and absence of holoenzyme and IHF. Footprints with UV light were made on 5-BrdU-substituted DNA and DNase I and laser UV footprints on conventional DNA templates. Results establish that the consensus thymidine residues of the UAS motif 5'-TGT are in close proximity to NifA. Reactivity suggests that each UAS thymidine is not structurally equivalent. Titration of NifA binding to the UAS in the presence or absence of the closed promoter complex indicates that the interaction of NifA with the UAS is not strongly co-operative with holoenzyme or IHF, a result supportive of an activation mechanism not reliant upon simple recruitment of factors to the promoter. Laser footprints demonstrated that holoenzyme suppressed reactivity of promoter consensus -14, -15 and -16 T residues, indicating close contact. Binding of holoenzyme resulted in a specific increase in 5-BrdU reactivity at -9 within the holoenzyme binding site, likely reflecting DNA distortion. Enhanced -9 reactivity required sigmaNN-terminal sequences that are necessary for activation. Since T-9 is melted in open complexes the closed complex appears poised for melting. Open promoter complex formation was accompanied by a distinct change in laser footprint signal at -11, consistent with the view that nucleation of strand separation occurs within or close to the -12 promoter element.

DNA bending and the initiation of transcription at 54-dependent bacterial promoters

Article

Full-text available

Oct 1997

We have examined the effects on transcription initiation of promoter and enhancer strength and of the curvature of the DNA separating these entities on wild-type and mutated enhancer-promoter regions at the Escherichia coli sigma54-dependent promoters glnAp2 and glnHp2 on supercoiled and linear DNA. Our results, together with previously reported observations by other investigators, show that the initiation of transcription on linear DNA requires a single intrinsic or induced bend in the DNA, as well as a promoter with high affinity for sigma54-RNA polymerase, but on supercoiled DNA requires either such a bend or a high affinity promoter but not both. The examination of the DNA sequence of all nif gene activator- or nitrogen regulator I-sigma54 promoters reveals that those lacking a binding site for the integration host factor have an intrinsic single bend in the DNA separating enhancer from promoter.

Casaz, P. & Buck, M. Probing the assembly of transcription initiation complexes through changes in N protease sensitivity. Proc. Natl. Acad. Sci. USA 94, 12145−12150

Article

Full-text available

Nov 1997

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.

Amino-terminal sequences of sigmaN (sigma54) inhibit RNA polymerase isomerization

Article

Full-text available

Mar 1999
GENE DEV

In bacteria, association of the specialized sigmaN protein with the core RNA polymerase subunits forms a holoenzyme able to bind promoter DNA, but unable to melt DNA and initiate transcription unless acted on by an activator protein. The conserved amino-terminal 50 amino acids of sigmaN (Region I) are required for the response to activators. We have used pre-melted DNA templates, in which the template strand is unpaired and accessible for transcription initiation, to mimic a naturally melted promoter and explore the function of Region I. Our results indicate that one activity of Region I sequences is to inhibit productive interaction of holoenzyme with pre-melted DNA. On pre-melted DNA targets, either activation of sigmaN-holoenzyme or removal of Region I allowed efficient formation of complexes in which melted DNA was sequestered by RNA polymerase. Like natural pre-initiation complexes formed on conventional DNA templates through the action of activator, such complexes were heparin-resistant and transcriptionally active. The inhibitory sigmaN Region I domain functioned in trans to confer heparin sensitivity to complexes between Region I-deleted holoenzyme and pre-melted promoter DNA. Evidence that Region I senses the conformation of the promoter was obtained from protein footprint experiments. We suggest that one function for Region I is to mask a single-strand DNA-binding activity of the holoenzyme. On the basis of extended DNA footprints of Region I-deleted holoenzyme, we also propose that Region I prevents RNA polymerase isomerization, a conformational change necessary for access to and the subsequent stable association of holoenzyme with melted DNA.

Bacterial sigma factors

Article

Jan 1992

The RpoN-box of the RNA polymerase sigma factor σN plays a role in promoter recognition

Article

Dec 1996

The RNA polymerase sigma factor σN (σ54) is characterized by the presence, near the C-terminal end of the protein, of a highly conserved sequence of 10 amino acids (ARRTVAKYRE) that has been termed the RpoN box. In order to examine the function of this motif, which is predicted to adopt an α-helical stucture, we have isolated a number of mutations that alter residues within the box and examined the properties of the σN derivatives encoded by them. Certain mutations that alter charged and potentially exposed residues within the motif result in transcriptionally inactive proteins with impaired promoter recognition but no impairment in core RNA polymerase binding. We therefore suggest that the RpoN box could play a direct or indirect role in recognition of the −24, −12 promoter consensus that is characteristic of σN-dependent genes.

Direct binding of transcription factor σ54 to promoter specific elements.

Conference Paper

Jan 1993

Active recruitment of σ54-RNA polymerase to the Pu promoter of Pseudomonas putida: Role of IHF and αCTD

Article

Sep 1998
EMBO J

The sequence elements determining the binding of the 54-containing RNA polymerase (54-RNAP) to the Pu promoter of Pseudomonas putida have been examined. Contrary to previous results in related systems, we show that the integration host factor (IHF) binding stimulates the recruitment of the enzyme to the -12/-24 sequence motifs. Such a recruitment, which is fully independent of the activator of the system, XylR, requires the interaction of the C-terminal domain of the subunit of RNAP with specific DNA sequences upstream of the IHF site which are reminiscent of the UP elements in 70 promoters. Our data show that this interaction is mainly brought about by the distinct geometry of the promoter region caused by IHF binding and the ensuing DNA bending. These results support the view that binding of 54-RNAP to a promoter is a step that can be subjected to regulation by factors (e.g. IHF) other than the sole intrinsic affinity of 54-RNAP for the -12/-24 site.

Protein–Protein Contacts that Activate and Repress Prokaryotic Transcription

Article

Mar 1998
CELL

Recently the structures of two of the DNA-binding domains of RNAP, the α-CTD (7xJeon, Y.H, Negishi, T, Shirakawa, M, Yamazaki, T, Fujita, N, Ishihama, A, and Kyogoku, Y. Science. 1995; 270: 1495–1497Crossref | PubMedSee all References, 6xGaal, T, Ross, W, Blatter, E.E, Tang, H, Jia, X, Krishnan, V.V, Assa-Munt, N, Ebright, R.H, and Gourse, R.L. Genes Dev. 1996; 10: 16–26Crossref | PubMedSee all References), and a portion of σ70 containing the −10 region recognition motif (Malhotra et al. 1996xMalhotra, A, Severinova, E, and Darst, S.A. Cell. 1996; 87: 127–136Abstract | Full Text | Full Text PDF | PubMed | Scopus (239)See all ReferencesMalhotra et al. 1996), have been determined. Such advances in the understanding of RNAP structure should facilitate elucidation of the more complex activation and repression mechanisms.In the case of σ70, this structural information in conjunction with the finding that σ plays an important role in directing and stabilizing promoter melting is likely to shed light on the mechanism of action of at least some activators that mediate their effects through σ. Since −10 region recognition involves base-specific contacts between the σ subunit and the melted nontemplate strand (Roberts and Roberts 1996xRoberts, C.W and Roberts, J.W. Cell. 1996; 86: 495–501Abstract | Full Text | Full Text PDF | PubMed | Scopus (107)See all ReferencesRoberts and Roberts 1996), it is possible that regulators that interact with σ may, in some cases, stabilize a conformation that favors the formation of these contacts, rather than merely stabilizing the binding of the −35 region recognition domain. Unfortunately, there is as of yet no high resolution structural information about the portion of σ that binds the promoter −35 region, the apparent target of a number of activators that bind in the immediate vicinity of the −35 box. Whether or not the effects of such activator–σ interactions can be transmitted through the structure of σ to the −10 region recognition motif remains to be learned.As the structural analysis of RNAP proceeds, it will be particularly informative to study complexes containing a DNA-bound regulator together with a relevant portion of RNAP. Finally, structural information about the catalytic subunits of RNAP are likely to enhance our understanding of how some activators that contact these subunits work.

Amino-terminal sequences of σN (σ54) inhibit RNA polymerase isomerization

Article

Feb 1999
GENE DEV

In bacteria, association of the specialized σN protein with the core RNA polymerase subunits forms a holoenzyme able to bind promoter DNA, but unable to melt DNA and initiate transcription unless acted on by an activator protein. The conserved amino-terminal 50 amino acids of σN (Region I) are required for the response to activators. We have used pre-melted DNA templates, in which the template strand is unpaired and accessible for transcription initiation, to mimic a naturally melted promoter and explore the function of Region I. Our results indicate that one activity of Region I sequences is to inhibit productive interaction of holoenzyme with pre-melted DNA. On pre-melted DNA targets, either activation of σN-holoenzyme or removal of Region I allowed efficient formation of complexes in which melted DNA was sequestered by RNA polymerase. Like natural pre-initiation complexes formed on conventional DNA templates through the action of activator, such complexes were heparin-resistant and transcriptionally active. The inhibitory σN Region I domain functioned in trans to confer heparin sensitivity to complexes between Region I-deleted holoenzyme and pre-melted promoter DNA. Evidence that Region I senses the conformation of the promoter was obtained from protein footprint experiments. We suggest that one function for Region I is to mask a single-strand DNA-binding activity of the holoenzyme. On the basis of extended DNA footprints of Region I-deleted holoenzyme, we also propose that Region I prevents RNA polymerase isomerization, a conformational change necessary for access to and the subsequent stable association of holoenzyme with melted DNA.

Structure of Transcription Elongation Complexes in Vivo

Article

Mar 1992

The opening of duplex DNA in the elongation phase of transcription by Escherichia coli RNA polymerase in vivo was detected at a regulatory site where a prolonged pause in transcription occurs. Single-stranded DNA in the transcription bubble was identified by its reactivity with potassium permanganate (KMnO4). The elongation structure in vivo was similar to that of transcription complexes made in vitro with some differences. The observed reactivity to KMnO4 of the DNA template strand was consistent with the existence of an RNA-DNA hybrid of about 12 nucleotides.

Specific binding of the transcription factor sigma-54 to promoter DNA

Article

Aug 1992

A central event in transcription is the assembly on DNA of specific complexes near the initiation sites for RNA synthesis. Activation of transcription by one class of enhancer-binding proteins requires an RNA polymerase holoenzyme containing the specialized transcription factor, sigma-54 (sigma 54). We report here that sigma 54 alone specifically binds to promoter DNA and is responsible for many of the close contacts between RNA polymerase holoenzyme and promoter DNA, a property proposed for the major sigma 70 protein family. Binding of sigma 54 to promoter DNA is not equivalent to that of holoenzyme suggesting that there is a constraint on sigma 54 conformation when bound with core RNA polymerase. Footprints indicate sigma 54 is at the leading edge of DNA-bound holoenzyme. Like the holoenzyme, sigma 54-binding to promoter DNA does not result in DNA strand separation. Instead the specific DNA-binding activity of sigma 54 assists assembly of a closed promoter complex. This complex can be isomerized to the open (DNA melted) complex by activator protein, but promoter-bound sigma 54 alone cannot be induced to melt DNA. The pathway leading to productive transcription is similar to that proposed for eukaryotic RNA polymerase II systems.

The phosphorylated form of the enhancer-binding protein NTRC has an ATPase activity that is essential for activation of transcription

Article

Nov 1991
CELL

The NTRC protein of enteric bacteria is an enhancer-binding protein that activates transcription in response to limitation of combined nitrogen. NTRC activates transcription by catalyzing formation of open complexes by RNA polymerase (sigma 54 holoenzyme form) in an ATP-dependent reaction. To catalyze open complex formation, NTRC must be phosphorylated. We show that phosphorylated NTRC has an ATPase activity, and we present biochemical and genetic evidence that NTRC must hydrolyze ATP to catalyze open complex formation. It is likely that all activators of sigma 54 holoenzyme have an ATPase activity.

Control site location and transcriptional regulation in Escherichia coli

Article

Oct 1991
Microbiol Rev

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.

DNA-looping and enhancer activity: association between DNA-bound NTRC activator and RNA polymerase at the bacterial glnA promoter

Article

Aug 1990

The NtrC protein activates transcription of the glnA operon of enteric bacteria by stimulating the formation of stable "open" complexes by RNA polymerase (sigma 54-holoenzyme form). To regulate the glnA promoter, NtrC binds to sites that have the properties of transcriptional enhancers: the sites will function far from the promoter and in an orientation-independent fashion. To investigate the mechanism of enhancer function, we have used electron microscopy to visualize the interactions of purified NtrC and RNA polymerase with their DNA binding sites and with each other. Under conditions that allow the formation of open complexes, about 30% of DNA molecules carry both RNA polymerase and NtrC bound to their specific sites. Of these, about 15% form looped structures in which NtrC and the RNA polymerase-promoter complex are in contact. The length of the looped DNA is that predicted from the spacing that was engineered between the enhancer and the glnA promoter (390 base pairs). As expected for activation intermediates, the looped structures disappear when RNA polymerase is allowed to transcribe the DNA. We conclude that the NtrC enhancer functions by means of a direct association between DNA-bound NtrC and RNA polymerase (DNA-looping model). Association of DNA-bound proteins appears to be the major mechanism by which different types of site-specific DNA transactions are localized and controlled.

Role of eukaryotic-type functional domains found in the prokaryotic enhancer receptor factor σ54

Article

Oct 1990
CELL

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.

Function of a Bacterial Activator Protein That Binds to Transcriptional Enhancers

Article

Mar 1989

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.

In vivo studies on the interaction of RNA polymerase-??54 with the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters: the role of NIFA in the formation of an open promoter complex

Article

Dec 1989

Transcription from the Klebsiella pneumoniae and Rhizobium meliloti nifH promoters requires the positive control protein NifA and the alternative sigma factor sigma 54, encoded by the rpoN gene. Transcription from the K. pneumoniae nifH promoter is fully dependent upon NifA bound at the upstream activator sequence (UAS) whereas the R. meliloti nifH promoter can be efficiently activated in the absence of this sequence and can also be activated by a mutant form of NifA unable to bind the UAS. The in vivo interaction of RNA polymerase-sigma 54 with these promoters was examined using dimethyl sulphate footprinting. The R. meliloti nifH promoter but not the K. pneumoniae nifH promoter showed sigma 54-dependent methylation protection of guanine residues at -14, -25 and -26, the most conserved nucleotides characteristic of sigma 54-dependent promoters. A mutant derivative of the K. pneumoniae nifH promoter bearing transitions at positions from -15 to -17 showed sigma 54-dependent methylation protection of guanines -13, -24 and -25. The enhanced interaction of the RNA polymerase-sigma 54 with this mutant promoter correlates with its increased level of activation by a form of NifA unable to bind the UAS. Use of in vivo KMnO4 footprinting to detect single-stranded pyrimidine residues and in vivo methylation protection demonstrated that the sigma 54-dependent protection observed in the R. meliloti and mutant K. pneumoniae nifH promoter results from the formation of a closed promoter complex. The isomerization of the pre-existing closed complex to an open promoter form, as judged by the local denaturation of promoter DNA which rendered sequences from +5 to -10 reactive towards KMnO4, was shown to be fully dependent on NifA. We propose a model in which the fidelity of activation of sigma 54-dependent promoters relies on a weak activator-independent interaction of RNA polymerase-sigma 54 with the promoter. A specific interaction of the appropriate activator with its respective UAS is then required for the positive control protein to facilitate open complex formation.

Protein Kinase and Phosphoprotein Phosphatase Activities of Nitrogen Regulatory Proteins NTRB and NTRC of Enteric Bacteria: Roles of the Conserved Amino-Terminal Domain of NTRC

Article

Aug 1988

The NTRC protein (ntrC product) of enteric bacteria activates transcription of nitrogen-regulated genes by a holoenzyme form of RNA polymerase that contains the ntrA product (sigma 54) as sigma factor. Although unmodified NTRC will bind to DNA, it must be phosphorylated to activate transcription. Both phosphorylation and dephosphorylation of NTRC occur in the presence of the NTRB protein (ntrB product). We here demonstrate rigorously that it is the NTRB protein that is a protein kinase by showing that NTRB can phosphorylate itself, whereas NTRC cannot. Phosphorylated NTRC (NTRC-P) is capable of autodephosphorylation with a first-order rate constant of 0.14-0.19 min-1 (t 1/2 of 5.0-3.6 min) at 37 degrees C. In addition, there is regulated dephosphorylation of NTRC-P. By contrast to the autophosphatase activity, regulated dephosphorylation requires three components in addition to NTRC-P: the PII regulatory protein, NTRB, and ATP. NTRC is phosphorylated within its amino-terminal domain, which is conserved in one partner of a number of two-component regulatory systems in a wide variety of eubacteria. A purified amino-terminal fragment of NTRC (approximately equal to 12.5 kDa) is sufficient for recognition by NTRB and is autodephosphorylated at the same rate as the native protein.

Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter

Article

Jul 1986
CELL

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.

Initiation of transcription at the bacterial glnAp2 promoter by purified E. coli components is facilitated by enhancers

Article

Oct 1987
CELL

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.

Changes in the DNA structure of the lac UV5 promoter during formation of an open complex with Escherichia coli RNA polymerase

Article

May 1985

By chemical and enzymatic methods, two stable complexes between Escherichia coli RNA polymerase and a linear DNA fragment carrying the lac UV5 promoter have been identified. In these binary complexes, DNA can adopt two alternate conformations as a function of temperature. Contacts between RNA polymerase and the DNA phosphate backbone are indistinguishable in these two forms, as revealed by probing with pancreatic DNase I. Protection of enhancement of the reactivity of the bases toward (CH3)2SO4 occurs, however, only in the form that predominates above 22 degrees C, RPo. The form stable at low temperature, RPi, is a "closed" complex since no single-stranded region is detectable in the DNA. The strong temperature dependence of the equilibrium constant, the midpoint value of the transition, and the rate of conversion between these two forms are in close agreement with a series of measurements performed by using a transcriptional assay and reported in the preceding paper [Buc, H., & McClure, W. R. (1985) Biochemistry (preceding paper in this issue)]. These data further support the postulated mechanism of open complex formation involving three sequential steps: R + P in equilibrium RPc in equilibrium RPi in equilibrium RPo. The binary complex RPc, which accumulates transiently at 37 degrees C before the isomerization leading to open complex formation, is not significantly protected against enzymatic cleavage or chemical modification and is therefore distinct from RPi and RPo.(ABSTRACT TRUNCATED AT 250 WORDS)

Converting Escherichia coli RNA Polymerase into an Enhancer-Responsive Enzyme: Role of an NH2-Terminal Leucine Patch in sigma54

Article

Dec 1995

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.

The bacterial enhancer-binding protein NTRC is a molecular machine: ATP hydrolysis is coupled to transcriptional activation

Article

Sep 1995
GENE DEV

NTRC is a prokaryotic enhancer-binding protein that activates transcription by sigma 54-holoenzyme. NTRC has an ATPase activity that is required for transcriptional activation, specifically for isomerization of closed complexes between sigma 54-holoenzyme and a promoter to open complexes. In the absence of ATP hydrolysis, there is known to be a kinetic barrier to open complex formation (i.e., the reaction proceeds so slowly that the polymerase synthesizes essentially no transcripts even from a supercoiled template). We show here that open complex formation is also thermodynamically unfavorable. In the absence of ATP hydrolysis the position of equilibrium between closed and open complexes favors the closed ones. Use of linear templates with a region of heteroduplex around the transcriptional start site--"preopened" templates--does not bypass the requirement for either NTRC or ATP hydrolysis, providing evidence that the rate-limiting step in open complex formation does not lie in DNA strand denaturation per se. These results are in contrast to recent findings regarding the ATP requirement for initiation of transcription by eukaryotic RNA polymerase II; in the latter case, the ATP requirement is circumvented by use of a supercoiled plasmid template or a preopened linear template.

Core RNA Polymerase and Promoter DNA Interactions of Purified Domains of σN: Bipartite Functions

Article

Jun 1995

The sigma N class of sigma factors confer upon RNA polymerase the requirement for enhancer-binding activator proteins. The sigma-N (sigma N) protein of Klebsiella pneumoniae was analysed by the assay of purified peptides comprising domains or regions of sigma N defined by proteolysis or by homology alignment, respectively. The NH2-terminal Region I is required for the correct interaction of holoenzyme with the promoter, and promoter complexes forming with a truncated sigma N lacking Region I are not activatable. The complexes lack the DNA structure believed to represent nucleated strand separation but still make close contacts with this promoter part. Determinants of specific DNA recognition by sigma N were shown to reside in a C-terminal 16 kDa peptide, and core RNA polymerase binding determinants in an adjacent peptide. The latter contacts and appears to pack against the DNA-binding domain. Thus the DNA-binding and core-binding domains are bipartite in function, consistent with core functioning as an allosteric effector of the sigma DNA-binding activity. The DNA-binding and core-binding domains together include Region III of sigma N. Although not the primary determinant of core or DNA recognition, the acidic Region II of sigma N influenced both activities. Regions I and II in combination with core RNA polymerase thus appear to control the activity of C-terminal DNA contacting surfaces to allow formation of a closed promoter complex that is susceptible to activation.

In a class of its own - The RNA polymerase sigma factor σ54 (σN)

Article

Jan 1994

Mike Merrick

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.

Promoter structure, promoter recognition, and transcription activation in prokaryotes

Article

Jan 1995
CELL

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The Domain Structure of Sigma 54 as Determined by Analysis of a Set of Deletion Mutants

Article

Mar 1994

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.

RNA polymerase binding using a strongly acidic hydrophobic-repeat region of σ54

Article

Apr 1994

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.

Activation and repression of E. coli promoters

Article

Nov 1996
CURR OPIN GENET DEV

Jay D. Gralla

There is no organism in which transcription initiation is better understood than Escherichia coli. Recent studies using genetics, biochemistry and structure analysis have revealed how RNA polymerase interactions at promoters are regulated. Prominent examples include the recruitment of polymerase by activators touching its alpha and sigma subunits; which subunit is touched depends on which activator is used and where it binds the DNA. The less-common cases centering on enhancer-dependent transcription use an entirely different mechanism, involving either DNA looping or tracking.

The RpoN-box motif of the RNA polymerase sigma factor σ(N) plays a role in promoter recognition

Article

Jan 1997

The RNA polymerase sigma factor sigma N (sigma 54) is characterized by the presence, near the C-terminal end of the protein, of a highly conserved sequence of 10 amino acids (ARRTVAKYRE) that has been termed the RpoN box. In order to examine the function of this motif, which is predicted to adopt an alpha-helical structure, we have isolated a number of mutations that alter residues within the box and examined the properties of the sigma N derivatives encoded by them. Certain mutations that alter charged and potentially exposed residues within the motif result in transcriptionally inactive proteins with impaired promoter recognition but no impairment in core RNA polymerase binding. We therefore suggest that the RpoN box could play a direct or indirect role in recognition of the -24, -12 promoter consensus that is characteristic of sigma N-dependent genes.

DNA-binding determinants of sigma 54 as deduced from libraries of mutations

Article

Mar 1997

PCR mutagenesis was used to obtain libraries of mutations in the region between amino acids 300 and 400 in the DNA-binding domain of Escherichia coli sigma 54. Two hundred changes that did not alter function were identified. These were compared with a somewhat smaller number of changes that did alter function. Several important regions were identified. Single point mutations in two of these, near amino acids 363 and 383, destroyed the ability of sigma to bind DNA, as assayed by band shift analysis. A third segment from amino acids 327 to 347 is also a candidate for contributing to DNA binding. Comparison with data in the literature leads to testable proposals for the complex mode of DNA binding that is associated with sigma 54.

Isolation and properties of enhancer-bypass mutants of sigma 54

Article

Apr 1997

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.

DNA-melting at the Bacillus subtilis flagellin promoter nucleates near −10 and expands unidirectionally

Article

Apr 1997

A central step in promoter activation by RNA polymerase (RNAP) is the localized separation of the DNA strands to form the transcription bubble. We have used potassium permanganate footprinting to monitor DNA strand-separation by the Bacillus subtilis sigmaD RNAP at the strong promoter, Phag, directing transcription of flagellin. The susceptibility of individual thymine bases to permanganate oxidation is influenced by temperature, Mg2+, nucleotides, and the RNAP delta subunit. In the absence of delta, sigmaD RNAP establishes a partially opened complex even at 0 degrees C with permanganate reactivity localized between -11 and -4 (RP(-4)). The region of strand separation expands to near -1 at 20 degrees C (RP(-1)) and to +3 at 40 degrees C (RP(+3)). The delta subunit inhibits the downstream propagation of the transcription bubble and thereby increases the concentration of early intermediates in the melting pathway. Indeed, E delta sigmaD forms a distinct nucleated complex (RPn) at 0 degrees C with a structural distortion localized to an AT base step within the -10 element. We propose a model for promoter melting in which strand separation nucleates within the conserved -10 consensus and subsequently propagates downstream. Mg2+ and nucleoside triphosphates (NTPs) favor the downstream propagation of the transcription bubble and strongly stimulate the RP(-1) to RP(+3) conversion. The NTP effects are apparently mediated by binding of substrate to the initiating NTP site: purines are more effective than pyrimidines and GMP alone can greatly increase the level of DNA-melting. The binding of substrates, but not Mg2+ alone, can effectively overcome the anti-melting effect of delta.

Promoter Recognition As Measured by Binding of Polymerase to Nontemplate Strand Oligonucleotide

Article

Jun 1997

In transcription initiation, the DNA strands must be separated to expose the template to RNA polymerase. As the closed initiation complex is converted to an open one, specific protein-DNA interactions involving bases of the nontemplate strand form and stabilize the promoter complex in the region of unwinding. Specific interaction between RNA polymerase and the promoter in Escherichia coliwas detected and quantified as the binding affinity of nontemplate oligonucleotide sequences. The RNA polymerase subunit sigma factor 70 contacted the bases of the nontemplate DNA strand through its conserved region 2; a mutation that affected promoter function altered the binding affinity of the oligonucleotide to the enzyme.

Multiple pathways to bypass the enhancer requirement of sigma 54 RNA polymerase: Roles for DNA and protein determinants

Article

Oct 1997

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.

Molecular Genetics of the RNA Polymerase II General Transcription Machinery

Article

Jul 1998

M Hampsey

Transcription initiation by RNA polymerase II (RNA pol II) requires interaction between cis-acting promoter elements and trans-acting factors. The eukaryotic promoter consists of core elements, which include the TATA box and other DNA sequences that define transcription start sites, and regulatory elements, which either enhance or repress transcription in a gene-specific manner. The core promoter is the site for assembly of the transcription preinitiation complex, which includes RNA pol II and the general transcription fctors TBP, TFIIB, TFIIE, TFIIF, and TFIIH. Regulatory elements bind gene-specific factors, which affect the rate of transcription by interacting, either directly or indirectly, with components of the general transcriptional machinery. A third class of transcription factors, termed coactivators, is not required for basal transcription in vitro but often mediates activation by a broad spectrum of activators. Accordingly, coactivators are neither gene-specific nor general transcription factors, although gene-specific coactivators have been described in metazoan systems. Transcriptional repressors include both gene-specific and general factors. Similar to coactivators, general transcriptional repressors affect the expression of a broad spectrum of genes yet do not repress all genes. General repressors either act through the core transcriptional machinery or are histone related and presumably affect chromatin function. This review focuses on the global effectors of RNA polymerase II transcription in yeast, including the general transcription factors, the coactivators, and the general repressors. Emphasis is placed on the role that yeast genetics has played in identifying these factors and their associated functions.

A bacterial ATP-dependent, enhancer binding protein that activates the housekeeping RNA polymerase

Article

Jul 1998
GENE DEV

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.

Promoter opening via a DNA fork junction binding activity

Article

Oct 1998

The rate-limiting step in transcriptional initiation typically is opening the promoter DNA to expose the template strand. Opening is tightly regulated, but how it occurs is not known. These experiments identify an activity, recognition of specific DNA fork junctions, and suggest that it is critical to bacterial promoter opening. This activity is both sequence and structure specific; it recognizes the bases that constitute the upstream double-stranded/single-stranded boundary of the open complex. Promoter mutations known to reduce opening rates lead to comparable reductions in fork junction binding affinity. The activity acts to establish the upstream boundary of melted DNA and works in conjunction with two single-stranded DNA binding activities that recognize separately the two melted strands. The junction binding activity is contained within the sigma factor component of the holoenzyme. The activity occurs in both a typical prokaryotic transcription system and in a eukaryotic-like bacterial system that responds to enhancers and needs ATP. Thus DNA opening catalyzed by fork junction binding may occur in a variety of systems in which DNA must be opened to be copied.

Multiple In Vivo Roles for the −12-Region Elements of Sigma 54 Promoters

Article

Dec 1998

Alignment of sigma 54-dependent promoters indicates conservation of two sequence elements. Six nucleotides in the downstream -12 element were mutated individually to each nonconsensus nucleotide. mRNA levels were measured in vivo for each promoter under strongly activating conditions. The results showed that the consensus sequence was not the strongest promoter. Instead, the -12 consensus element consists of two subregions that behave differently when mutated. Single changes in the upstream TTT consensus subregion can lead to increases in transcription, whereas single changes in the downstream GC(A/T) can lead to decreases in transcription. Selected double mutations with changes in both subregions were constructed and studied in vivo. No double mutation increased promoter strength, and some decreased it. Mutant promoters were also assayed under nonactivating conditions in vivo. No mRNA was detected in 23 of the 24 promoters tested. However, one double mutant showed substantial levels of transcript, indicating that the -12 sequence was capable of specifying basal transcription under nonactivating conditions. Overall, the results show that the -12 region has multiple roles in transcription in vivo, including modulating both basal and induced RNA levels.

Identification of an N-Terminal Region of Sigma 54 Required for Enhancer Responsiveness

Article

Dec 1998

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.

Region I modifies DNA-binding domain conformation of sigma 54 within the holoenzyme

Article

Feb 1999

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.

A fork junction DNA-protein switch that controls promoter melting by the bacterial enhancer-dependent sigma factor

Abstract

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