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

Siva R. Wigneshweraraj, Konstantin KuznedelovDagger ||, Konstantin SeverinovDagger §, and Martin Buck

From the Department of Biological Sciences, Imperial College of Science, Technology and Medicine, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ, United Kingdom and the Dagger  Waksman Institute and Department of Genetics, Rutgers, The State University, Piscataway, New Jersey 08904

Received for publication, September 16, 2002, and in revised form, October 17, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 2beta beta 'omega ; E) associates with a sigma (sigma ) subunit to form the holoenzyme (Esigma ). 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 Esigma 54 regulatory center at the start site proximal promoter element where activator binds and DNA melting originates.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Multisubunit DNA-dependent RNA polymerases (RNAP)1 are complex molecular machines that synthesize a RNA copy from a DNA template. In Escherichia coli, five subunits (alpha 2beta beta 'omega ) form the RNAP catalytic core (E) that associates with a sigma (sigma ) subunit to form the holoenzyme (Esigma ). The sigma  subunit imparts on the core RNAP the ability to specifically recognize and initiate transcription from promoters. Biochemical and structural studies indicate that the protein-protein contacts at the interface between core RNAP and sigma , an extensive and functionally specialized sets of surfaces, govern the conformational changes that allow efficient promoter recognition and transcription initiation (1-3).

Sequence comparisons reveal two unrelated families of RNAP sigma  factors. Members of the major family of sigma  factors form RNAP holoenzymes that recognize promoters and form transcriptionally competent promoter complexes in the absence of other factors or energy sources. This family, which includes most bacterial sigma  factors is named after the prototypical housekeeping sigma  of E. coli, sigma 70. Members of the second, minor family of sigma  factors, the sigma 54 family, form RNAP holoenzymes that recognize promoters but require additional protein factors and a source of energy in the form of ATP or GTP hydrolysis for formation of transcriptionally competent promoter complexes (4-6). Despite the differences in pathways that lead to transcription-competent open promoter complexes, both classes of sigma  factors occupy similar positions within their respective RNAP holoenzymes and appear to utilize some common RNAP surfaces for transcription initiation (7-10).

Binding of a sigma 70 family factor induces conformational changes within the core RNAP (2, 11, 12). Structural modules of the core RNAP, designated as the beta ' clamp, the beta  flap, and the beta  lobes, interact with a sigma 70 family sigma  subunit (sigma A) in the structures of Thermus aquaticus and Thermus thermophilus RNAP holoenzymes and undergo conformational changes, which orientate and position sigma 70 DNA-binding domains within the RNAP holoenzyme to allow promoter recognition (2, 3). The importance of these conformational changes is underlined by our recent observation that removal of the E. coli RNAP beta  flap domain abolished the ability of the mutant Esigma 70 to recognize promoters of the -10/-35 class (13).

Esigma 54 recognizes and binds promoters containing conserved consensus elements centered around -24 and -12 nucleotides upstream of the transcription start site at +1. These promoter elements can be considered as functional analogues of the -35/-10 consensus promoter elements recognized by Esigma 70 class holoenzymes. The activity of sigma 54 promoters is strictly regulated at the DNA melting step and is dependent upon the presence of an enhancer DNA-bound activator. The maintenance of the transcriptionally silent state of the activator-independent Esigma 54 closed complex depends on the integrity of (i) the amino-terminal 56 amino acids of sigma 54 (known as Region I; Fig. 1a) and (ii) promoter sequences at -12 (14-17). In the closed complex, Region I localizes close to the -12 promoter element where DNA melting originates. This protein-DNA arrangement, which we called the "regulatory center," constitutes a target for mechanochemical action of the activator (18, 19). Very little is known about the contribution of the core RNAP mobile modules to promoter binding and transcription initiation by Esigma 54. Here, we studied the properties of E. coli Esigma 54 reconstituted from mutant core RNAP harboring the beta  flap deletion, Delta 885-914 (hereafter called Delta flapE), to gain insights into the contribution of a core RNAP structural module, which is critical for transcription initiation by Esigma 70, to enhancer-dependent transcription by Esigma 54 (Fig. 1b). Our results demonstrate that the beta  flap domain of the RNAP has multiple roles in transcription by the Esigma 54. Most importantly, it appears to orchestrate the formation and organization of the regulatory center at the start site proximal promoter element at -12.


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Fig. 1.   a, domain organization of K. pneumoniae sigma 54. b, domain organization of the E. coli beta  subunit (top) and structure of the T. aquaticus core RNAP (bottom). In the E. coli beta  subunit, the gray shaded regions indicate evolutionarily conserved segments (A-I). Dispensable regions are shown in white. Evolutionarily conserved segment G is expanded and aligned with corresponding segments from T. aquaticus (Taq) and yeast RNAP II (YP2). Dots and hyphens indicate identical or missing amino acids, respectively. The secondary structure of the beta  flap from T. aquaticus is also given. The deletion mutation (Delta 885-914) characterized in this work is shown above the E. coli sequence. In the T. aquaticus core RNAP structure, the beta , beta ', alpha 2, and omega  are shown in cyan, pink, green, and white, respectively. The active center Mg2+ is shown in blue. The portion of the beta  flap deleted in this work is shown in yellow. c, S. meliloti nifH 88-mer homo- and heteroduplex promoter probes used in this work. The consensus GG and GC elements of sigma 54-dependent promoters are in bold and their positions with respect to the transcription start site at +1 is given. Boxed are the sequences that are mismatched to generate the early and late melted heteroduplex probes.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteins and Promoter DNA Probes

Klebsiella pneumoniae sigma 54, mutant variants thereof (Delta R1sigma 54, Ala24-26, R336Asigma 54, Cys20sigma 54, and Cys46sigma 54), E. coli full-length PspF and PspF-(1-275) were purified as amino-terminal His6-tagged fusion proteins essentially as described in Refs. 16, 18, 20, and 21.

Wild-type and mutant E. coli RNAP core enzymes, which contained a COOH-terminal hexahistidine tag on the plasmid-borne beta  subunit, were purified as follows. Plasmid pRL706 expressing wild-type rpoB, or the corresponding plasmid expressing mutant rpoB with the flap deletion were transformed in the E. coli XL1-Blue cells and cells were grown in 4 liters of LB containing 200 µg/ml ampicillin. Cells were grown to A600 of ~2 without induction. Cells (~14 g) were collected and lysed, by sonication, in 70 ml of grinding buffer (40 mM Tris-HCl, pH 7.9, 10 mM EDTA, 15 mM beta -mercaptoethanol, and 0.2 mM phenylmethylsulfonyl fluoride) containing 200 mM NaCl. The supernatant after low speed centrifugation was made 0.8% with Polymin P (Sigma), pH 8.0. The Polymin P pellet was washed twice with grinding buffer containing 500 mM NaCl, and RNAP was eluted twice with 15 ml of grinding buffer containing 1000 mM NaCl. The combined 1000 mM extract was precipitated with ammonium sulfate (0.3 g/ml), the pellet was collected, dissolved in 40 ml of TG buffer (40 mM Tris-HCl, pH 7.9, 5% glycerol), and loaded onto a 5-ml Heparin HiTrap column (Amersham Biosciences) equilibrated in TG buffer containing 100 mM NaCl. The column was washed with TG buffer containing 300 mM NaCl, and RNAP was eluted in TG buffer containing 600 mM NaCl. The 600 mM NaCl fraction was loaded onto a 5-ml HiTrap chelating column (Amersham Biosciences) loaded with Ni2+ using the manufacturer's instructions and equilibrated in a buffer containing 25 mM HEPES, pH 8.0, 500 mM NaCl, 5% glycerol. The column was step-eluted with the same buffer containing 10, 20, 50, and 200 mM imidazole, pH 8.0. RNAP containing plasmid-borne, hexahistidine-tagged mutant beta  eluted at 50 mM imidazole in the buffer and did not contain sigma 70 subunit as judged by visual inspection of Coomassie-stained SDS gels and abortive initiation assays on a strong sigma 70-dependent promoter. RNAP was precipitated by ammonium sulfate, the pellet was dissolved in 250 µl of TG buffer containing 1 mM EDTA and loaded onto a Superose-6 column (Amersham Biosciences) equilibrated in TG buffer containing 1 mM EDTA and 200 mM NaCl. The column was developed isocratically with the same buffer, RNAP-containing fractions were collected, concentrated on Centricon-100 (Amicon) to ~0.3 mg/ml, glycerol was added to a final concentration of 10%, the enzyme was aliquoted and stored at -80 °C. For native PAGE and footprinting assays, 32P end-labeled 88-mer (from -60 to +26) homoduplex and heteroduplex Sinorhizobium meliloti nifH promoter probes were prepared essentially as described by Cannon et al. (21).

Core RNAP Binding Assays

Native Gel Assembly Assay-- Increasing amounts of core RNAP (10-400 nM) was added to 10-µl reactions containing 100 nM 32P end-labeled sigma 54 in core binding buffer (40 mM Tris-Cl, pH 8, 10% (v/v) glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl) at 37 °C. The reactions were incubated for 5 min and complexes were resolved on a 4.5% native polyacrylamide gel. RNAP holoenzyme complexes were visualized and quantified by PhosphorImager analysis of the dried gel.

RNAP Holoenzyme Dissociation Assays-- For core RNAP-sigma 54 dissociation assays, 50 nM holoenzyme complexes (at a molar ratio of 1:6 core RNAP to unlabeled sigma 54) were pre-formed in core binding buffer containing 200 µg/ml alpha -lactoalbumin in a total volume of 50 µl and incubated for 5 min at 37 °C. After incubation, 300 nM 32P-labeled sigma 54 was added for 0-60 min and samples were taken and loaded on a 4.5% native polyacrylamide gel that was run for 2 h at 50 V. PhosphorImager analysis was used to quantify 32P-labeled sigma 54·RNAP complexes and the free sigma 54 in each sample.

Promoter Probe Binding Assays

These were conducted essentially as described by Wigneshweraraj et al. (10) in STA buffer (25 mM Tris acetate, pH 8.0, 8 mM magnesium acetate, 10 mM KCl, 1 mM dithiothreitol, and 3.5% (w/v) PEG 8000) using 100 nM sigma 54-RNAP (formed with 1:6 ratio of core RNAP to sigma 54), 16 nM 32P end-labeled homoduplex or heteroduplex (as indicated in figures and legends thereof) S. meliloti nifH promoter probes, 4 mM ATP or GTP (where indicated), and 4 µM PspF-(1-275) (where indicated) at 37 °C. When required heparin (100 µg/ml) was added for 5 min prior to analysis of the complexes by native PAGE. Gels were dried and promoter complexes were quantified by PhosphorImager analysis.

Activator Binding Assays

Activator binding assays were conducted as described by Chaney et al. (18). Holoenzymes were formed by incubating 50 nM 32P-labeled sigma 54 with 100 nM core RNAP at 37 °C in STA buffer for 5 min. 10 µM PspFDelta HTH, 0.2 mM ADP, and 5 mM sodium fluoride were added to the reaction for a further 5 min; 0.2 mM aluminum chloride was then added and the reactions were further incubated for 10 min at 37 °C prior to separation of complexes by native PAGE. Dried gels were visualized using a PhosphorImager.

DNA Footprinting Assays

DNase I Footprinting-- DNase I footprinting of closed, open, and initiated promoter complexes formed on the S. meliloti nifH homoduplex (Fig. 4a) or early melted (Fig. 8c) promoter probes (32P end-labeled template strand) were conducted essentially as described by Wigneshweraraj et al. (10). The 10-µl binding reactions were conducted in STA buffer; 1.75 × 10-3 units of DNase I (Amersham Biosciences) was added (for 1 min), reactions terminated, and bound and unbound DNAs were separated by native PAGE. Unbound and RNAP-bound DNA was then excised from the gels. Gel-isolated DNA was eluted into 0.1 mM EDTA overnight at 37 °C and recoveries of the isolated DNA were determined by dry Cherenkov counting. Equal numbers of counts were loaded onto a 10% denaturing gel. Dried gels were visualized and quantified by using a PhosphorImager.

KMnO4 Footprinting-- Binding reactions were conducted as described above for DNase I footprinting in STA buffer without dithiothreitol. 4 mM fresh KMnO4 was added for 30 s, followed by 50 mM beta -mercaptoethanol to quench DNA oxidation. The reactions were phenol:chloroform:isoamyl alcohol extracted, ethanol precipitated, and stored overnight at -80 °C. The DNA was then pelleted and washed with 80% (v/v) ethanol. The dried DNA pellet was resuspended in 50 µl of TE buffer (10 mM Tris-Cl, pH 7.0, and 0.1 mM EDTA) to which 1 µl of 0.4% (w/v) SDS and 500 µl of a 25 mg/ml stock of proteinase K were added and incubated for 30 min at 37 °C. The reaction was stopped by phenol:chloroform:isoamyl alcohol extraction and the DNA was precipitated and pelleted as before. The DNA pellet was then resuspended in 30 µl of H2O and the oxidized DNA was cleaved with 10% (v/v) piperidine at 90 °C for 20 min. The cleavage reaction was stopped by flash-freezing. The reaction was then dried using a speed-vacuum drier. The recoveries of DNA was quantified and analyzed by denaturing PAGE as described above for DNase I footprinting.

Ortho-Copper Phenanthroline (Ortho-CuOP) Footprinting-- Binding reactions were conducted essentially as described for KMnO4 footprinting. The reactions were treated with 0.5 µl of a solution of 4 mM ortho-phenanthroline and 0.92 mM CuSO4 followed by 0.5 µl of 0.116 M mercaptopropionic acid for 2 min. The reaction was terminated by the addition of 1 µl of 28 mM 2,9-dimethyl-1,10-phenanthroline and loaded onto a 4.5% native gel. Unbound and RNAP bound DNA was then excised from the gels. Gel-isolated DNA was eluted into H2O overnight at 37 °C and processed and analyzed on a 10% denaturing gel as described above.

In Vitro Transcription Assays

These were performed essentially as described by Wigneshweraraj et al. (10). For the single round transcription assay, supercoiled plasmid (pMKC28 or pSLE1) containing the S. meliloti nifH or the E. coli pspA promoter, respectively, was used. Plasmids pMKC28 and pSLE1 contain a T7 early transcriptional terminator sequence downstream of the multiple cloning site. The promoter fragment is inserted into the multiple cloning site in such a way to direct transcription to generate a discrete transcript of ~470 (for pMKC28) and ~510 (for pSLE1) bases. 10-µl reactions contained 20 nM template, 100 nM RNAP holoenzyme (formed with 1:6 ratio of core RNAP to sigma 54). Where indicated 4 mM ATP or GTP and 4 µM PspF-(1-275) were added for open complex formation. The elongation mixture contained 100 µg/ml heparin, 0.1 mM ATP, CTP, and GTP, and 1.5 µCi of [alpha -32P]UTP. Reactions were done at 37 °C and stopped with 4 µl of formamide dye mixture. 7 µl of the samples were run on a 6% denaturing gel and the dried gel was quantified and analyzed by PhosphorImager analysis. Run-off transcription assays were performed using the late melted promoter probe essentially as described above. However, reactions were stopped by phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation. Recoveries of transcripts were determined by dry Cherenkov counting and equal numbers of counts were loaded onto 15% denaturing gels. Dried gels were visualized and quantified by using a PhosphorImager.

FeBABE Cleavage of Promoter Complexes

Single-cysteine forms of sigma 54, 20sigma 54, and 46sigma 54 were modified with the FeBABE reagent ((p-bromoacetamidobenzyl)-EDTA Fe; Dojindo Chemicals) and conjugation yield determined essentially as described by Wigneshweraraj et al. (9). DNA cleavage assays were performed as described by Wigneshweraraj et al. (19). Briefly, promoter complexes were formed (in 10-µl reactions) using 20 nM S. meliloti nifH early melted promoter probe (template strand 32P end-labeled), 50 nM core RNAP, and 300 nM sigma 54 in cleavage buffer (40 mM HEPES, pH 8.0, 10 mM MgCl2, 5% (v/v) glycerol, 0.1 mM KCl, and 0.1 mM EDTA) at 37 °C. DNA cleavage was initiated by the rapid sequential addition of 2 mM sodium ascorbate, pH 7.0, and 1 mM hydrogen peroxide. Reactions were allowed to proceed for 10 min and quenched with 50 µl of stop buffer (0.1 M thiourea and 100 µg/ml sonicated salmon sperm DNA). The stopped reactions were phenol:chloroform:isoamyl alcohol extracted and precipitated with ethanol. Recoveries of DNA were determined by dry Cherenkov counting and equal numbers of counts were loaded onto 10% denaturing gels. Dried gels were visualized and analyzed using a PhosphorImager.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Core RNAP Lacking the beta  Flap Forms an Unstable sigma 54 Holoenzyme

Previously, we have demonstrated that deletion of the beta  flap does not significantly affect the ability of mutant core RNAP to bind sigma 70 (13). We tested the ability of Delta flapE to bind sigma 54. For this purpose, a fixed amount of 32P-labeled sigma 54 (32P-sigma 54) was combined with various amounts of Delta flapE or control wild-type core RNAP and the mixtures were separated on a native polyacrylamide gel (Fig. 2a, (i)) and quantified using a PhosphorImager (Fig. 2a (ii)) (10, 20). As shown in Fig. 2a, with the wild-type RNAP most of the 32P-sigma 54 was in the holoenzyme form (Esigma 54) when the molar ratio of core RNAP to sigma 54 reached 1:1. In contrast, a 4-fold excess of Delta flapE was required to convert 32P-sigma 54 to Delta flapEsigma 54. Thus, it appears that Delta flapE has a reduced ability to bind sigma 54, suggesting that the beta  flap contributes to the sigma 54 binding site.


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Fig. 2.   Binding of Delta flap core RNAP to sigma 54. a: i, native gel showing the binding of 32P-sigma 54 to wild-type and Delta flap core RNAP. The migration positions of 32P-sigma 54 and RNAP holoenzyme are indicated. ii, quantification of the data from i using a PhosphorImager. Saturation curves for Delta flapEsigma 54 (black-triangle) and Esigma 54 (black-square) formation are shown. Indicated on the x axis is the molar ratio of core RNAP to 32P-sigma 54 included in the reaction and on the y axis the percentage of RNAP holoenzyme formation. b, stability of Delta flapEsigma 54 and Esigma 54 over time (1-60 min; shown on the top of the gel). Lanes 1, 3, 5, 7, 9, and 11 contain the wild-type RNAP; lanes 2, 4, 6, 8, 10, and 12 contain the Delta flap RNAP.

To estimate the stability of the Delta flapEsigma 54 complex, we conducted experiments in which unlabeled sigma 54 was mixed with core RNAP at a ratio of 6:1 to allow complete conversion of mutant core RNAP into the holoenzyme form, and then added 32P-sigma 54 to capture free core RNAP arising from dissociation of the preformed holoenzyme complex. As shown in Fig. 2b, little formation of the 32P-labeled RNAP holoenzyme was detected 1 min after the addition of 32P-sigma 54 to the wild-type RNAP holoenzyme (lane 1), indicating that the wild-type Esigma 54 complex had not yet dissociated. Strikingly, ~6-fold increased formation of 32P-labeled RNAP holoenzyme complex was observed in the case of Delta flapEsigma 54 (Fig. 2b, lane 2), indicating that the mutant holoenzyme dissociated much faster than did the wild-type Esigma 54. The presence of promoter DNA did not markedly improve the stability of Delta flapEsigma 54 (data not shown). Overall, the data suggests that the beta  flap contributes to the binding of sigma 54 to RNAP core and the stability of the resulting RNAP holoenzyme. This observation is consistent with previous results in which a derivative of sigma 54 harboring a FeBABE at residue Cys198 within the major core RNAP binding surface of sigma 54 (residues 120-215; Fig. 1a) cleaved the RNAP beta  subunit between residues 850 and 890 (9, 20), close to or within the beta  flap domain (residues 885-914).

The beta  Flap Contributes to Heparin-stable Promoter Complex Formation by Esigma 54

In Esigma 70, sigma 70 region 4 (which recognizes the -35 promoter consensus element) interacts with the beta  flap (2, 13). This interaction is functionally important and is required for correct positioning of the sigma 70 region 4 to allow promoter complex formation on promoters of the -35/-10 class (2, 13). To investigate the contribution of the beta  flap domain to promoter binding by Esigma 54, we analyzed promoter complexes formed by Delta flapEsigma 54 on the S. meliloti nifH promoter and its derivatives (Fig. 1c) under activating and nonactivating conditions.

Experiments with S. meliloti nifH Homoduplex Promoter Probe-- Initially, Delta flapEsigma 54 binding activity was investigated on a fully double-stranded S. meliloti nifH promoter probe (homoduplex probe; Fig. 1c). Complex formation was investigated under three conditions that allowed the monitoring of different promoter complexes. Under nonactivating conditions, when RNAP alone or with added ATP and GTP was present in the reaction, only closed promoter complexes were expected to form. Under activating conditions, when RNAP, E. coli sigma 54-dependent activator PspF-(1-275) (a form of PspF that lacks the enhancer DNA binding domain)2 and ATP were present, open promoter complexes were expected to form. Under initiating conditions, when RNAP, PspF-(1-275), and GTP were present, RNAP could use GTP to synthesize an RNA trimer.

Wild-type or mutant Esigma 54 were used to form promoter complexes that were then resolved by native gel electrophoresis, or were first challenged with heparin, followed by electrophoretic separation of promoter complex from free promoter probe. Acquisition of heparin stability by Esigma 54 promoter complex on the homoduplex promoter probe is a hallmark of either open or initiated Esigma 54 promoter complexes. The results were quantified and are presented in Table I. Under nonactivating conditions Delta flapEsigma 54 bound the homoduplex probe with similar activity as Esigma 54, and the complexes were sensitive to heparin, as expected. Promoter complex formed by the wild-type Esigma 54 became heparin-stable under activating or initiating conditions, as expected. In contrast, neither activation nor initiation conditions led to the acquisition of heparin stability by Delta flapEsigma 54 on the homoduplex promoter (Table I). Because in the absence of heparin Delta flapEsigma 54 and Esigma 54 bound the homoduplex probe equally well, we conclude that (i) the beta  flap contributes to DNA opening and/or engagement of single-stranded DNA by Esigma 54 to acquire heparin stability, or (ii) deletion of the beta  flap compromises the ability of the mutant RNAP to respond to activation conditions.

                              
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Table I
Activities of the wild-type Esigma 54 and Delta flapEsigma 54 to bind and form heparin stable promoter complexes on the S. meliloti nifH homoduplex promoter probe and heteroduplex variants thereof

Experiments with S. meliloti nifH Heteroduplex Promoter Probes-- To distinguish between the above possibilities, we performed binding and heparin stability assays on a heteroduplex promoter probe. The late-melted promoter probe contains a mismatched segment from positions -10 to -1 relative to the transcription start point and is believed to represent the conformation of promoter DNA in Esigma 54 open complexes (21, 22) (Fig. 1c). Esigma 54 efficiently forms heparin-stable promoter complexes on the late-melted promoter probe only under activating conditions. Initial binding assays with Delta flapEsigma 54 in the absence of heparin showed that Delta flapEsigma 54 bound the late-melted probe with wild-type activity under all the conditions tested (Table I). Like Esigma 54, Delta flapEsigma 54 was unable to form heparin-stable complexes in the absence of activation (Table I). Activation (+PspF-(1-275) and ATP) increased the heparin stability of wild-type Esigma 54. In contrast, activated Delta flapEsigma 54 late melted promoter DNA complexes were heparin-sensitive (Table I). However, under activating conditions that permitted initiation (+PspF-(1-275) and GTP) a significant number of heparin-stable Delta flapEsigma 54 promoter complexes was detected (Table I), suggesting that (i) Delta flapEsigma 54 was able to respond to activation when the template strand was available as single-stranded DNA, and (ii) initiation of RNA synthesis stabilized Delta flapEsigma 54 on the late melted promoter probe.

Acquisition of heparin stability on late melted promoter probes independent of activation is a property of deregulated forms of Esigma 54, for example, those lacking the sigma 54 regulatory Region I (Delta RIsigma 54) (21) (Fig. 1a). To further assess the ability of Delta flapEsigma 54 to engage pre-melted promoter DNA in a heparin-stable manner we conducted experiments with double mutant holoenzyme lacking the beta  flap and sigma 54 regulatory Region I. Initially, we tested whether Delta RIsigma 54 can form holoenzyme complexes with Delta flapE. The results showed that a 1:8 ratio of Delta flapE to Delta RIsigma 54 was needed to form stable Delta flapDelta RIsigma 54 complexes, consistent with the sigma 54 binding defects of Delta flapE (data not shown and Fig. 2). In the absence of heparin, Delta flapDelta RIsigma 54 bound the late melted promoter probe with an activity similar to that of EDelta ·RIsigma 54 (data not shown). Strikingly and in marked contrast to EDelta ·RIsigma 54 complexes, very little (<2%) of the Delta flapDelta RIsigma 54 late melted promoter complexes survived heparin challenge (Fig. 3, compare lane 2 with 5). However, conditions that permitted activator-independent initiation by EDelta ·RIsigma 54 (i.e. the presence of GTP) detectably stabilized Delta flapDelta RIsigma 54-late melted promoter complexes (Fig. 3, compare lane 5 with 6). In contrast, the presence of noninitiating nucleotides, ATP or dGTP, did not lead to heparin resistance of Delta flapDelta RIsigma 54 promoter complexes (data not shown). We note that increased heparin resistance of Delta flapDelta RIsigma 54 promoter complexes was not dependent on the presence of activator (Fig. 3, compare lanes 6 and 7), suggesting that the deregulated properties of RIsigma 54 have not been altered in the absence of the beta  flap. Similar binding patterns were obtained with Delta flapE in the context of other deregulated mutants of sigma 54, such as F318A (23), R336A (24), and Ala24-26sigma 54 (16), further confirming that initiation of RNA synthesis stabilizes late melted promoter complexes in the context of Delta flap RNAP (data not shown).


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Fig. 3.   Heparin stability of EDelta R1sigma 54 and Delta flapDelta R1sigma 54 on the S. meliloti nifH late melted promoter probe. Samples were taken following a 5-min heparin challenge and analyzed by native-PAGE. The percentage of late melted promoter probe complexes that survive the heparin challenge is indicated. The migration positions of promoter complexes and unbound DNA are also shown.

Overall, several conclusions can be derived from the binding properties of Delta flapEsigma 54 on the late melted promoter probe. First, DNA interactions with melted DNA made by Esigma 54 are destabilized in the absence of the beta  flap. Second, Delta flapEsigma 54 closed complexes respond to activator and engage pre-melted DNA to form a heparin-stable complex, albeit less efficiently than the wild-type Esigma 54. This result suggests that the beta  flap is not essential to the RNAP site that interacts with the activator. Third, the deletion of the beta  flap does not deregulate Esigma 54, at least in the context of the late melted promoter probe complex.

DNase I Footprinting Reveals Altered Interactions of Delta flapEsigma 54 with the S. meliloti nifH Promoter

To further characterize the marked differences in promoter complex stability caused by deletion of the beta  flap we conducted DNase I footprinting experiments. Promoter complexes on homoduplex DNA were formed in the absence of heparin because of the instability of mutant open and initiated complexes (Table I), footprinted, and complexes were separated from free DNA by native PAGE. Both Esigma 54 and Delta flapEsigma 54 formed equal amounts of promoter complexes under the conditions tested (data not shown). Promoter DNA from promoter complexes was recovered and analyzed by denaturing PAGE (see "Experimental Procedures"). The DNase I footprint of the Esigma 54 on the S. meliloti nifH homoduplex probe was typical of Esigma 54 closed promoter complexes. Promoter DNA was protected from DNase I cleavage between positions -34 and -5 with respect to the transcription start site at +1 (Fig. 4a (i), lane 3). The Delta flapEsigma 54 closed promoter complex footprint was similar to that of wild-type Esigma 54 between positions -34 and -4 (Fig. 4a (i), lanes 3 and 4, and (ii)). PhosphorImager analysis of the wild-type and mutant closed promoter complex footprints revealed that several sites on the DNA outside of the closed complex-specific protection region (-34 to -4) were also protected from DNase I cleavage in the mutant closed promoter complexes (Fig. 4a (ii), indicated by arrows). This result is suggestive of an overall inhibition of DNase I cleavage, perhaps because of (i) altered conformation of the Delta flapEsigma 54 closed promoter complex or (ii) nonspecific binding of the Delta flapEsigma 54. In promoter complexes formed with the wild-type Esigma 54, open promoter complex formation or the initiation of RNA synthesis results in the extension of the DNase I footprint in the downstream direction (Fig. 4a (i), compare lane 3 with lanes 6 and 8, respectively). No further increased protection of downstream DNA was detected in open complexes formed with the Delta flapEsigma 54 (Fig. 4a (i), compare lanes 4 and 5). Strikingly, conditions that allowed initiation from Delta flapEsigma 54 promoter complexes resulted in increased DNase I cleavage between -34 and -5 and shortening of the footprint in the upstream direction (Fig. 4a (i), lane 7). Analysis of mutant-initiated complexes by native PAGE revealed that the Delta flapEsigma 54 formed an equal number of initiated promoter complexes as Esigma 54, suggesting that the increased cleavage of DNA between the -34 and -5 positions was not because of simple dissociation of promoter complexes (data not shown and Table I). Overall, the DNase I footprinting data suggests that Delta flapEsigma 54 promoter complexes differ significantly from promoter complexes formed by Esigma 54. The failure to observe a fully extended downstream footprint in mutant complexes under activating conditions, as well as the apparent hypersensitivity of mutant initiated complexes to DNase I possibly indicates an altered response of the Delta flapEsigma 54 closed complexes to activation and altered conformations of Delta flapEsigma 54 compared Esigma 54 under closed complex conditions.


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Fig. 4.   a, i, DNase I footprints of closed, open, and initiated promoter complexes promoter complex formed by Esigma 54 and Delta flapEsigma 54 on the S. meliloti nifH homoduplex promoter probe. The solid and dotted lines represent closed complex specific and open/initiated complex specific extension of the footprints, respectively. ii, PhosphorImager traces of lanes 3 and 4 from i. As before, the solid and dotted lines indicate closed complex specific footprint and activator-dependent extension of the footprints, respectively. Arrows indicate prominent sites outside the closed complex specific protection region that show reduced sensitivity to DNase I cleavage in Delta flapEsigma 54 closed promoter complexes. b, PhosphorImager traces of Esigma 54 (black lines) and Delta flapEsigma 54 (gray lines) closed, open, and initiated complexes after probing with KMnO4.

Delta flapEsigma 54 Is Defective for Promoter DNA Melting

We used DNA melting within open or initiated promoter complexes as an indicator of activator responsiveness and analyzed promoter complexes formed by Delta flapEsigma 54 and the wild-type Esigma 54 by KMnO4 probing. Reactions were set up essentially as described above for DNase I footprinting. As shown in Fig. 4b, in the absence of activation, DNA melting was barely detectable within closed promoter complexes formed by the Delta flapEsigma 54. However, very little deregulated DNA melting was observed within the wild-type closed promoter complex. Activation of Esigma 54 promoter complexes resulted in increased KMnO4 reactivity of template strand thymines at positions -10 and -8 indicating promoter opening in response to activation (Fig. 4b, (ii)). In contrast, the KMnO4 reactivity of the -10 and -8 thymines was greatly reduced within activated mutant promoter complexes. Under initiating conditions, a 2-fold further increase in KMnO4 reactivity of thymine at position -8 was seen in wild-type promoter complexes, consistent with the expectation that initiation stabilizes DNA opening (Fig. 4b, compare (ii) and (iii)). In contrast, no significant increase in KMnO4 reactivity was detected in Delta flapEsigma 54-initiated promoter complexes. Overall, KMnO4 probing data are consistent with DNase I footprinting results and strongly suggests that Delta flapEsigma 54 promoter complexes are defective for (i) DNA melting and/or (ii) activator responsiveness.

Delta flapEsigma 54 Forms a Stable Complex with the sigma 54 Activator PspF-(1-275)

To determine whether the absence of the beta  flap affects the ability of Esigma 54 to form a stable binary complex with sigma 54 activator PspF-(1-275), we conducted binding assays in the presence of ADP-AlFx, a nonhydrolysable analogue of ATP that allows stable activator-Esigma 54 complexes to form (18). Esigma 54 or Delta flapEsigma 54 were combined with PspF-(1-275) in the presence of ADP-AlFx and protein complexes were analyzed by native PAGE. Fig. 5 shows that both holoenzymes formed similar amounts of stable, ADP-AlFx-dependent complexes with PspF-(1-275) (Fig. 5, compare lane 3 with 6). Similar results were obtained in the presence of promoter DNA (data not shown). Thus, it appears that the greatly reduced DNA melting in Delta flapEsigma 54 promoter complexes formed under activating or initiating conditions is not because of a major defect in activator interaction. Furthermore, these activator binding assays also suggest that the beta  flap is not part of the putative beta  subunit activator interaction site previously detected by chemical cross-linking (25).


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Fig. 5.   Native gel showing the nucleotide (ADP-AlFx)-dependent binding of PspF-(1-275) to Esigma 54 and Delta flapEsigma sigma 54. The migration positions of sigma 54, Esigma 54, Delta flapEsigma 54, and activator complexes thereof are indicated. Lanes 1-3 contain the wild-type Esigma 54 and lanes 4-6 contain the Delta flapEsigma 54. RNAP holoenzymes were formed using 32P-sigma 54.

Delta flapEsigma 54 Is Defective for Activator-dependent in Vitro Transcription from Supercoiled Templates

To assess the consequence of sigma 54-binding and promoter DNA-interaction defects of Delta flapE on transcription activity, we conducted single-round in vitro transcription assays using the supercoiled plasmid pMKC28, which contains the S. meliloti nifH promoter (24). When ATP and PspF-(1-275) were used to form open complexes prior to the addition of heparin, [alpha -32P]UTP, and the remaining nucleotides, Delta flapEsigma 54 was only about 15% as active as the wild-type Esigma 54 for transcription (Fig. 6, compare lanes 1 and 2). This result is very consistent with the results of the late melted promoter complex stability assay where only about 14% of the initial Delta flapEsigma 54 promoter complexes survived heparin challenge (Table I). When GTP and PspF-(1-275) were used to form initiated promoter complexes prior to the addition of heparin and remaining nucleotides, Delta flapEsigma 54 showed less than 5% of the wild-type Esigma 54 activity, indicating a destabilization of complexes under initiating conditions. This result is striking as the presence of GTP-stabilized Delta flapEsigma 54 and Delta flapEDelta R1sigma 54 complexes on late melted promoter (Table I and Fig. 3, respectively). However, the result is consistent with the DNase I and KMnO4 footprinting data, where initiation resulted in an altered DNase I footprint for Delta flapEsigma 54 (Fig. 4a (i), lane 7) and greatly reduced DNA melting was observed in mutant complexes at initiating conditions (Fig. 4b, (iii)). Additional in vitro transcription assays using supercoiled pSLE1 plasmid, which harbors the pspA promoter and the DNA binding site for PspF, and full-length PspF (26), gave transcription patterns similar to those obtained with PspF-(1-275) (Fig. 6b, compare lanes 1 and 2 with 3 and 4), suggesting that the presence of enhancer-bound activator did not detectably affect transcription activity of the Delta flapEsigma 54. Overall, the in vitro transcription data from supercoiled templates show that Delta flapEsigma 54 is transcriptionally active, but to a lesser degree than Esigma 54. A likely explanation for this could be the weakened sigma 54 and promoter DNA interactions made by Esigma 54 in the absence of the beta  flap.


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Fig. 6.   Activator-dependent in vitro transcription activity of Esigma 54 and Delta flapEsigma 54 from supercoiled plasmid pMKC28 harboring the S. meliloti nifH promoter and PspF-(1-275) (a) and supercoiled plasmid pSLE1 harboring the pspA operon and full-length PspF (b). For a and b, the percentage of transcripts produced with respect to wild-type Esigma 54 is given. c, activator-dependent in vitro transcription activity of Esigma 54 and Delta flapEsigma 54 from the S. meliloti nifH late melted promoter probe. Lane 9 contains molecular weight markers and the activity of the Delta flapEsigma 54 is given with respect to wild-type Esigma 54 activity. The arrow indicates the ~28-bp transcript.

Delta flapEsigma 54 Is Active for Activator-dependent in Vitro Transcription from Heteroduplex Promoter Probes

To investigate whether by-passing the DNA melting step would permit efficient transcription by Delta flapEsigma 54, in vitro transcription from the S. meliloti nifH late melted promoter probe was performed. After closed promoter complex formation, PspF-(1-275) and either ATP or GTP were added to induce open or initiated promoter complex formation, respectively. Next, a mixture of heparin, [alpha -32P]UTP, and remaining nucleotides was added to allow a single round of transcription to occur. When ATP and PspF-(1-275) were used for activation, the Delta flapEsigma 54 was 40% as active as the wild-type Esigma 54 (Fig. 6c, compare lanes 3 and 4). This run-off transcript was of the expected size (~28 bp) and its production was sigma 54 and activator-dependent (Fig. 6c, lanes 1, 2 and 5, 6, respectively). In the presence of GTP and PspF-(1-275), transcription by the Delta flapEsigma 54 was poor, resulting in ~12% of wild-type activity. Delta flapEsigma 54 was inactive on the S. meliloti nifH homoduplex promoter probe (data not shown). Thus, it appears that some of the in vitro transcription defects of the Esigma 54 associated with the Delta flap mutation can be overcome by using pre-melted promoter templates. Overall, the single-round and run-off transcription data, together with the KMnO4 probing of mutant promoter complexes, strongly implies that the Delta flapEsigma 54 is defective for stable DNA melting in response to activation.

Delta flapEsigma 54 Is Inactive for in Vitro Transcription with Deregulated sigma 54 Mutants

In deregulated forms of Esigma 54 DNA opening occurs in the absence of activator, but the resultant open complexes are unstable unless initiated. As a result, transcription by deregulated Esigma 54 proceeds via an unstable open complex that was proposed to correspond to an intermediate promoter complex prior to fully formed stable open complex formation (27). Therefore, transcription by deregulated Esigma 54 measures the formation of unstable intermediate open complexes. We performed activator-independent transcription assays with three different deregulated mutants of sigma 54 (Delta RIsigma 54, Ala24-26sigma 54 and R336Asigma 54 in the context of the Delta flap RNAP holoenzyme. The activator-independent transcription assay was performed using the S. meliloti nifH promoter in the pMKC28 plasmid (24). To allow activator-independent initiation, RNAP holoenzymes, template DNA, and GTP were preincubated, followed by the addition of heparin to destroy residual unstable complexes. [alpha -32P]UTP and remaining nucleotides were next added to initiate a round of transcription. As expected, RNAP holoenzymes reconstituted from wild-type core RNAP and either of the three deregulated sigma 54 mutants were active in both activator-dependent (Fig. 7, lanes 2-4) and activator-independent (Fig. 7, lanes 6-8) transcription. Strikingly, the double mutant holoenzymes reconstituted from Delta flapE and either of the deregulated sigma 54 mutants were transcriptionally inactive either in the presence (Fig. 7, lanes 10-12) or absence (Fig. 7, lanes 14-16) of activator. This result contrasts the late melted promoter probe stability data in which the deregulated forms of Delta flapEsigma 54 formed heparin-stable complexes in the presence of GTP independent of activation (Fig. 3 and data not shown). We therefore considered a possibility that deregulated forms of Delta flapEsigma 54 were defective for promoter DNA binding. However, DNase I footprints of closed complexes formed with deregulated forms of Delta flapEsigma 54 indicated that this was not the case (data not shown). Overall, it appears that removal of the beta  flap while only partially affecting the ability of wild-type sigma 54 RNAP holoenzyme to transcribe from supercoiled templates, completely abolishes transcription by RNAP holoenzymes reconstituted with deregulated forms of sigma 54. This suggests that the beta  flap is required for unstable open promoter complex formation and/or that the beta  flap is needed for transcription initiation from the unstable promoter complexes.


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Fig. 7.   Activator-independent in vitro transcription activity of deregulated forms of Esigma 54 and Delta flapEsigma 54 from the supercoiled plasmid pMKC28 containing the S. meliloti nifH promoter.

Delta flapEsigma 54 Makes Altered DNA Interactions within Closed Promoter Complexes

The apparently contradicting properties exhibited by deregulated forms of Delta flapEsigma 54 in the late melted promoter probe binding experiments and transcription assays prompted us to investigate DNA interactions made by Delta flapEsigma 54 early during open promoter complex formation. The transcriptionally silent state of the Esigma 54 closed promoter complexes strictly depends on the integrity of promoter sequences immediately downstream of the -12 promoter consensus element (the GC-element; Fig. 1c). In wild-type Esigma 54 closed promoter complexes, the base pair immediately downstream of the GC element shows increased reactivity toward KMnO4, diethylpyrocarbonate, and ortho-CuOP and therefore appears to be melted (28). This limited DNA opening within closed promoter complexes is termed "early DNA melting" and is one hallmark of regulated Esigma 54 transcription. In stable activator-dependent, transcriptionally competent open complexes, early DNA melting is not evident, suggesting that early DNA melting is a transient feature en route to regulated transcription by Esigma 54 (28). To investigate early DNA melting within closed promoter complexes formed by Delta flapEsigma 54, ortho-CuOP footprinting was performed. As expected, DNA cleavage by ortho-CuOP was seen at position -12 (immediately downstream the GC-element; Fig. 1c) in closed complexes formed with the wild-type Esigma 54 on the S. meliloti nifH homoduplex promoter probe (Fig. 8a, lane 3). Activation of the wild-type closed complexes resulted in ~5-fold reduction of the -12 signal, indicating open complex formation (Fig. 8a, compare lanes 3 and 5). In contrast, cleavage at -12 was absent in closed complexes formed by Delta flapEsigma 54 (Fig. 8a, lane 4), suggesting that the mutant enzyme is either defective for early DNA melting and/or is unable to make stable interactions with early melted DNA. To test the latter possibility, we conducted ortho-CuOP footprinting assays on promoter complexes formed on a heteroduplex promoter probe in which 2 base pairs immediately downstream of the GC element were mismatched, thus mimicking early promoter melting (Fig. 1c, early melted promoter probe). Ortho-CuOP treatment of early melted DNA in the absence of RNAP revealed a hypersensitive site around the mismatched region, as expected (Fig. 8b, compare lanes 1 and 2), and Esigma 54 protected the mismatched DNA region from ortho-CuOP cleavage, indicating the binding of Esigma 54 to the promoter probe (Fig. 8b, compare lanes 1 and 3). A clear protection of the hypersensitive site at -12 was also seen in reactions containing Delta flapEsigma 54 indicating complex formation by Delta flapEsigma 54 on the early melted promoter probe (Fig. 8b, lane 4). DNase I footprinting experiments also showed protection between DNA positions -34 to -5 in the presence of both Esigma 54 and Delta flapEsigma 54 (Fig. 8c, compare lanes 3 and 4), thus confirming efficient complex formation on early melted promoter probe by both enzymes.


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Fig. 8.   Ortho-copper phenanthroline (ortho-CuOP) footprints of Esigma 54 and Delta flapEsigma 54 promoter complexes formed on the S. meliloti nifH (a) homoduplex and (b) early melted promoter probes. The hypersensitive site at -12 is indicated. The dotted lines indicate the region of promoter that becomes protected upon Esigma 54/Delta flapEsigma 54 binding. c, DNase I footprints of Esigma 54 and Delta flapEsigma 54 promoter complexes formed on the S. meliloti nifH early melted promoter probe. The solid line indicates the region that becomes protected upon promoter complex formation by Esigma 54 and Delta flapEsigma 54, respectively.

To gain further insights into Delta flapEsigma 54 interaction with early melted DNA, we tested heparin stability of nifH promoter early melted complexes. As summarized in Table I, Delta flapEsigma 54 bound the early melted promoter probe as efficiently as wild-type Esigma 54 under all conditions tested. As reported previously (29, 30), and in contrast to the situation on homoduplex promoter probes, conditions that permitted activation (+PspF-(1-275) and ATP) or initiation (+PspF-(1-275) and GTP) did not markedly increase the heparin stability of wild-type Esigma 54 complexes on the early melted promoter probe (Table I). This property of wild-type Esigma 54 complexes is attributed to tight binding of sigma 54 to the heteroduplex region of the early melted promoter probe (15, 29). Significantly, the Delta flapEsigma 54-early melted promoter probe complex was unstable and did not survive heparin challenge under any of the conditions tested (Table I), suggesting that Esigma 54-early melted DNA interactions are altered in the absence of the beta  flap and do not lead to stable promoter complex formation. Alternatively, it is also possible that the mutant holoenzyme dissociates to core RNAP and free sigma  in the presence of heparin. However, band shift analysis of holoenzymes on native gels similar to the ones shown in Fig. 2 showed that Delta flapEsigma 54 was as sensitive to disruption by heparin as the wild-type Esigma 54 (data not shown). Thus, the failure of the Delta flapEsigma 54 to form heparin-stable complexes on the early melted promoter probe seems to be because of a failure of the Delta flap core RNAP to bring about a DNA-dependent change(s) in the core RNAP-sigma 54 interaction that results in the formation of heparin-stable promoter complexes. In other words, we suggest that Delta flapEsigma 54 is not defective in the binding to the early melted DNA structure, but is defective in the formation and maintenance of the early melted structure.

Organization of the Regulatory Center within Esigma 54 Closed Complexes Is Dependent on the beta  Flap

Regulated transcription by Esigma 54 relies on the integrity of sigma 54 Region I (Fig. 1a), which localizes proximal to promoter DNA around the GC promoter element (14-16, 19, 31-34) (Fig. 1c). Within the RNAP holoenzyme, sigma 54 Region I localizes proximal to the beta  and beta ' subunit residues that contribute to the formation of the RNAP active center (9). Region I is directly contacted by activators of Esigma 54 transcription (18). We have termed the sigma 54 Region I-DNA-core RNAP arrangement within closed Esigma 54 promoter complexes the regulatory center (19). Experiments presented above suggest that some of the properties of Delta flapEsigma 54 could be accounted for by defects in the organization of the regulatory center. To investigate whether the beta  flap contributes to proper positioning of sigma 54 Region I and hence to the organization of the regulatory center, we conducted localized hydroxyl radical cleavage experiments using FeBABE derivatives of sigma 54. RNAP holoenzymes were reconstituted with the wild-type and mutant RNAP core enzymes and sigma 54 containing FeBABE conjugated to Cys20 and Cys46 (20*sigma 54 and 46*sigma 54)3 in Region I4 and were used to form complexes on early melted promoter probes. Preliminary experiments demonstrated that the mutant and the wild-type holoenzymes reconstituted with 20*sigma 54 and 46*sigma 54 formed promoter complexes on the early melted promoter probe as efficiently as holoenzymes reconstituted with unmodified sigma 54 (data not shown). After complex formation, hydroxyl radicals were generated and the pattern of cleavage within the early melted promoter probe is presented in Fig. 9. All resulting cleavage of promoter DNA was dependent on the FeBABE reagent being coupled to unique cysteine residues in Region I because no cleavage was observed in control complexes formed with RNAP holoenzymes reconstituted with cysteine-free sigma 54 (Cys-freesigma 54) that was subjected to FeBABE conjugation conditions as a control (Fig. 9, lanes 1, 2, 7, and 8). In contrast, strong cleavage of promoter DNA template strands between positions -11 and -16 was seen within promoter complexes formed by E20*sigma 54 (Fig. 9, lanes 3 and 4), suggesting that sigma 54 Region I residue 20 is proximal to the regulatory center. Strikingly, no DNA cleavage was detected in complexes formed by Delta flapE20*sigma 54 (Fig. 9, compare lanes 3 and 4 with 9 and 10), suggesting that in the absence of the beta  flap, residue 20 in sigma 54 Region I is unable to occupy its correct position in the regulatory center. In E46*sigma 54 complexes, cleavages in the template strand of promoter DNA between positions -6 and -2 were obtained (Fig. 9, compare lanes 5 and 6), indicating that residue 46 in sigma 54 Region I is localized outside of the regulatory center. Interestingly, Delta flapE46*sigma 54 complexes also cleaved promoter DNA at these positions (Fig. 9, compare lanes 5 and 6 with 11 and 12), suggesting that the positioning of sigma 54 Region I residues that do not contribute to the regulatory center is not affected by the Delta flap mutation, at least in the context of this assay. Thus, this suggests that the beta  flap directly and selectively contributes to the organization of the Esigma 54 regulatory center.


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Fig. 9.   FeBABE footprints of S. meliloti nifH early melted promoter probe. FeBABE cleavage profiles of S. meliloti nifH template strand by 20*sigma 54, 46*sigma 54, and Cys-freesigma 54 in the context of the wild-type and Delta flap RNAP. Broken lines indicate the cluster of cleavage sites. Lanes M contain a mixture of the end-labeled S. meliloti nifH promoter DNA fragments as a molecular weight marker.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transcription from enhancer-dependent promoters relies upon the RNAP containing the sigma 54 factor. Unlike the enhancer-independent promoters recognized by the sigma 70 containing RNAP, the conversion of closed enhancer-dependent promoter complexes to transcription-competent open complexes requires the mechanochemical action of ATPases belonging to the AAA+ protein family (4, 35). The contribution of the RNAP mobile structural modules (beta  flap, beta ' clamp, beta  downstream and upstream lobes) to enhancer-dependent and enhancer-independent transcription by Esigma 54 and Esigma 70, respectively, is not well understood at a molecular level. By using a mutant RNAP harboring a deletion of the beta  flap (Delta 885-914; Delta flap), we have investigated the contribution of this RNAP structural module to Esigma 54 functioning. The data establish that the beta  flap domain has multiple roles in enhancer-dependent transcription. Strikingly, the beta  flap domain appears to have different functionalities in enhancer-dependent and enhancer-independent transcription.

sigma 54 Binding-- Even though the enhancer-dependent sigma 54 and the enhancer-independent sigma 70 bind the common RNAP with similar affinities and occupy similar positions within the RNAP holoenzyme (7, 9, 36), the Delta flap mutation markedly reduces the activity of the RNAP to bind sigma 54 and the stability of the resulting holoenzyme. In contrast, sigma 70 binding and the stability of Esigma 70 were not greatly affected by the Delta flap mutation (13). Holoenzyme formation is accompanied by large-scale conformational changes of all the structural modules of the beta  and beta ' subunits that serve to orientate and position the sigma  factor for promoter-specific transcription initiation. Upon binding sigma 70, the beta  flap re-positions region 4 of sigma 70 to facilitate recognition of the -35 promoter element (2, 3, 13). The contribution of the beta  flap to Esigma 54 formation appears to be 2-fold. First, the instability of the Delta flapEsigma 54 complexes suggests that the beta  flap contributes to the anchoring of sigma 54 to the core RNAP. Second, differences in the DNA cleavage patterns by FeBABE-modified sigma 54 in the context of the wild-type and Delta flap RNAP imply a role for the beta  flap in the proper positioning of sigma 54 domains within the holoenzyme that is important for Esigma 54 functioning.

Promoter Complex Formation-- Promoter complex formation by Esigma 70 induces major conformational changes in two of the mobile modules of the RNAP, notably also including the beta  flap domain (3). Consequently, Esigma 70 with the Delta flap mutation is unable to efficiently bind and utilize the -35/-10 class of bacterial promoters (13). In the context of the Esigma 54, the Delta flap mutation has little effect on initial promoter complex formation as judged by gel shift and DNase I footprinting assays. In contrast to Esigma 54, Delta flapEsigma 54 promoter complexes formed under activating conditions were sensitive to heparin and KMnO4 probing showed greatly reduced DNA opening within such mutant promoter complexes. Consequently, in vitro transcription activity of the Delta flapEsigma 54 from supercoiled templates is severely affected. By-passing the DNA melting step by using heteroduplex promoter probes marginally improves the transcription activity of the Delta flapEsigma 54. In contrast, DNA opening was fully normal in promoter complexes formed with the Delta flapEsigma 70, but mutant promoter complexes were less stable than the wild-type ones to heparin challenge.5

The isomerization of closed promoter complexes to open complexes proceeds via several heparin-sensitive intermediates involving several protein and DNA conformational changes and alterations in the interfaces between the RNAP and DNA (37). It is proposed that the entry of DNA into the active-cleft of the RNAP triggers a slow protein conformational change that nucleates DNA melting and the formation of the heparin-resistant open promoter complex (38). We propose that the low levels of open complex formation by the Delta flapEsigma 54 proceed via an altered pathway that neither results in efficient DNA melting nor acquisition of heparin stability. The limitation of the Delta flapEsigma 54 to only function with the wild-type sigma 54 for transcription, but not with deregulated sigma 54 mutants that transcribe via unstable and heparin-sensitive open complexes, supports this view. Thus, we propose that the beta  flap contributes to the formation of an intermediate, heparin-sensitive promoter complex state or states en route to heparin stable Esigma 54 open complex formation.

Acquisition of Heparin Stability-- On the late melted promoter probe, activated Esigma 54 promoter complexes are resistant to heparin, suggesting that the Esigma 54 must undergo conformational changes in response to activation to acquire stability (21, 22). The Delta flapEsigma 54 does not efficiently form heparin-stable complexes on the late-melted promoter under activating conditions, suggesting that, in the absence of the beta  flap, the Esigma 54 does not undergo the same set of conformational changes that leads to heparin stability. Furthermore, unlike the wild-type Esigma 54, the Delta flapEsigma 54 also forms heparin-sensitive complexes on the early melted promoter probe under nonactivating conditions. Thus, we propose that the beta  flap makes a significant contribution to the acquisition of heparin stability by the Esigma 54. Previously, we have reported that either the absence of sigma 54 Region I or mutations affecting the beta  subunit downstream lobe functionality reduces the ability of the Esigma 54 to form heparin-stable promoter complexes (10, 29). We envisage a functional co-operation between Region I of sigma 54 and two mobile structural modules, the beta  flap and the beta  downstream lobe, which leads to heparin stable-promoter complex formation during enhancer-dependent transcription.

Organization of the Esigma 54 Regulatory Center-- In enhancer-dependent closed complexes formed by the Esigma 54, Region I of sigma 54, and the catalytic center of the RNAP co-localize over the -12 position (GC-element) of the promoter where DNA melting originates. This nucleoprotein complex is defined as the Esigma 54 regulatory center and constitutes the target for the activator protein (9, 18, 19). Comparison of FeBABE cleavage profiles of closed promoter complexes formed with the wild-type and Delta flap RNAP clearly indicates that residues in Region I of sigma 54 that contribute to the regulatory center are not proximal to the regulatory center in the absence of the beta  flap domain. This suggests that the beta  flap may directly contribute to the formation of the regulatory center by positioning Region I over the GC-element. Furthermore, the FeBABE data also serves to help explain the absence of the Region I-dependent DNA distortion within closed complexes formed with the Delta flapEsigma 54 in ortho-CuOP footprinting experiments.

Overview-- Comparison of multisubunit RNAP structures of bacterial and yeast RNAPs and promoter complexes thereof reveals conserved structural mobile modules and conformational changes that are important for the functioning of the RNAP as a complex molecular machine (39). Factors that target these mobile modules act to modulate and regulate the activity of RNAPs. In bacteria, sigma  factors make extensive interactions with the structural mobile modules (beta ' clamp, beta  flap, and beta  downstream and upstream lobes) of the bacterial RNAP during the process of transcription initiation (1, 2, 40). Unlike the beta  downstream lobe module, which is commonly used by Esigma 54 and Esigma 70 during open complex formation (8, 10), the beta  flap module makes markedly different contributions to enhancer-dependent and -independent transcription by Esigma 54 and Esigma 70, respectively. For Esigma 70, the beta  flap plays a crucial role in the proper positioning of sigma 70 region 4 within the holoenzyme for promoter-specific transcription initiation (13). Thus, it appears that for enhancer-independent transcription by Esigma 70, the beta  flap contributes to RNAP-promoter DNA interactions at the start site distal promoter element. For Esigma 54, the beta  flap has multiple roles in modulating Esigma 54 activity and most important of these appears to be the positioning of Region I at the start site proximal promoter element. It remains to be determined whether it is sigma 54 Region I or other parts of sigma 54 that interact with the beta  flap en route to enhancer-dependent transcription initiation. However, the data presented here highlights the fact that the extensive interface between sigma  factor and RNAP is crucial for the co-ordinated reconfiguration of both partners for efficient transcription initiation.

    ACKNOWLEDGEMENT

We thank Wendy Cannon for comments on the manuscript.

    FOOTNOTES

* This work was supported by a Biotechnology and Biological Sciences Research Council project grant (to M. B.) and National Institutes of Health RO1 Grant GM64530 (to K. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence may be addressed. Tel.: 732-445-6095; Fax: 732-445-573; E-mail: severik@waksman.rutgers.edu.

To whom correspondence may be addressed. Tel.: 44-207-594-5442; Fax: 44-207-594-5419; E-mail: m.buck@ic.ac.uk.

|| On leave from Limnological Institute of the Russian Academy of Sciences, Irkutsk, Russia.

Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M209442200

2 E. coli PspF-(1-275) lacks the helix-turn-helix enhancer DNA binding domain and essentially represents the central catalytic domain of the protein.

3 Superscript asterisk indicates conjugation to FeBABE.

4 S. R. Wigneshweraraj, P. Burrows, K. Severinov, A. Ishihama, and M. Buck, manuscript in preparation.

5 K. Severinov and K. Kuznedelov, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: RNAP, RNA polymerase; CuOP, ortho-copper phenanthroline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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