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

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.

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 (␣ 2 ␤␤Ј) form the RNAP catalytic core (E) that associates with a sigma () subunit to form the holoenzyme (E). The 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 , an extensive and functionally specialized sets of surfaces, govern the conformational changes that allow efficient promoter recognition and transcription initiation (1)(2)(3).
Sequence comparisons reveal two unrelated families of RNAP factors. Members of the major family of 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 factors is named after the prototypical housekeeping of E. coli, 70 . Members of the second, minor family of factors, the 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 factors occupy similar positions within their respective RNAP holoenzymes and appear to utilize some common RNAP surfaces for transcription initiation (7)(8)(9)(10).
Binding of a 70 family factor induces conformational changes within the core RNAP (2,11,12). Structural modules of the core RNAP, designated as the ␤Ј clamp, the ␤ flap, and the ␤ lobes, interact with a 70 family subunit ( A ) in the structures of Thermus aquaticus and Thermus thermophilus RNAP holoenzymes and undergo conformational changes, which orientate and position 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 ␤ flap domain abolished the ability of the mutant E 70 to recognize promoters of the Ϫ10/Ϫ35 class (13). E 54 recognizes and binds promoters containing conserved consensus elements centered around Ϫ24 and Ϫ12 nucleotides upstream of the transcription start site at ϩ1. These promoter elements can be considered as functional analogues of the Ϫ35/Ϫ10 consensus promoter elements recognized by E 70 class holoenzymes. The activity of 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 E 54 closed complex depends on the integrity of (i) the amino-terminal 56 amino acids of 54 (known as Region I; Fig. 1a) and (ii) promoter sequences at Ϫ12 (14 -17). In the closed complex, Region I localizes close to the Ϫ12 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 E 54 . Here, we studied the properties of E. coli E 54 reconstituted from mutant core RNAP harboring the ␤ flap deletion, ⌬885-914 (hereafter called ⌬flap E), to gain insights into the contribution of a core RNAP structural module, which is critical for transcription initiation by E 70 , to enhancer-dependent transcription by E 54 (Fig. 1b). Our results demonstrate that the ␤ flap domain of the RNAP has multiple roles in transcription by the E 54 . Most importantly, it appears to orchestrate the formation and organization of the regulatory center at the start site proximal promoter element at Ϫ12.

Proteins and Promoter DNA Probes
Klebsiella pneumoniae 54 , mutant variants thereof (⌬R1 54 , Ala24 -26, R336A 54 , Cys 20 54 , and Cys 46 54 ), E. coli full-length PspF and PspF-(1-275) were purified as amino-terminal His 6 -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 ␤ 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 g/ml ampicillin. Cells were grown to A 600 of ϳ2 without induction. Cells (ϳ14 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 ␤-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 Ni 2ϩ 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 ␤ eluted at 50 mM imidazole in the buffer and did not contain 70 subunit as judged by visual inspection of Coomassie-stained SDS gels and abortive initiation assays on a strong 70 -dependent promoter. RNAP was precipitated by ammonium sulfate, the pellet was dissolved in 250 l 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 ϳ0.3 mg/ml, glycerol was added to a final concentration of 10%, the enzyme was aliquoted and stored at Ϫ80°C. For native PAGE and footprinting assays, 32 P end-labeled 88-mer (from Ϫ60 to ϩ26) 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-l reactions containing 100 nM 32 P end-labeled 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-54 dissociation assays, 50 nM holoenzyme complexes (at a molar ratio of 1:6 core RNAP to unlabeled 54 ) were pre-formed in core binding buffer containing 200 g/ml ␣-lactoalbumin in a total volume of 50 l and incubated for 5 min at 37°C. After incubation, 300 nM 32 P-labeled 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 32 P-labeled 54 ⅐RNAP complexes and the free 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 54 -RNAP (formed with 1:6 ratio of core RNAP to 54 ), 16 nM 32 P 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 M PspF-(1-275) (where indicated) at 37°C. When required heparin (100 g/ml) was added for 5 min prior to FIG. 1. a, domain organization of K. pneumoniae 54 . b, domain organization of the E. coli ␤ subunit (top) and structure of the T. aquaticus core RNAP (bottom). In the E. coli ␤ 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 ␤ flap from T. aquaticus is also given. The deletion mutation (⌬885-914) characterized in this work is shown above the E. coli sequence. In the T. aquaticus core RNAP structure, the ␤, ␤Ј, ␣ 2 , and are shown in cyan, pink, green, and white, respectively. The active center Mg 2ϩ is shown in blue. The portion of the ␤ 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 54 -dependent promoters are in bold and their positions with respect to the transcription start site at ϩ1 is given. Boxed are the sequences that are mismatched to generate the early and late melted heteroduplex probes. 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 32 P-labeled 54 with 100 nM core RNAP at 37°C in STA buffer for 5 min. 10 M PspF⌬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 ( 32 P endlabeled template strand) were conducted essentially as described by Wigneshweraraj et al. (10). The 10-l binding reactions were conducted in STA buffer; 1.75 ϫ 10 Ϫ3 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.
KMnO 4 Footprinting-Binding reactions were conducted as described above for DNase I footprinting in STA buffer without dithiothreitol. 4 mM fresh KMnO 4 was added for 30 s, followed by 50 mM ␤-mercaptoethanol to quench DNA oxidation. The reactions were phenol:chloroform:isoamyl alcohol extracted, ethanol precipitated, and stored overnight at Ϫ80°C. The DNA was then pelleted and washed with 80% (v/v) ethanol. The dried DNA pellet was resuspended in 50 l of TE buffer (10 mM Tris-Cl, pH 7.0, and 0.1 mM EDTA) to which 1 l of 0.4% (w/v) SDS and 500 l 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 l of H 2 O and the oxidized DNA was cleaved with 10% (v/v) piperidine at 90°C for 20 min. The cleavage reaction was stopped by flashfreezing. 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 KMnO 4 footprinting. The reactions were treated with 0.5 l of a solution of 4 mM ortho-phenanthroline and 0.92 mM CuSO 4 followed by 0.5 l of 0.116 M mercaptopropionic acid for 2 min. The reaction was terminated by the addition of 1 l 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 H 2 O 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 ϳ470 (for pMKC28) and ϳ510 (for pSLE1) bases. 10-l reactions contained 20 nM template, 100 nM RNAP holoenzyme (formed with 1:6 ratio of core RNAP to 54 ). Where indicated 4 mM ATP or GTP and 4 M PspF-  were added for open complex formation. The elongation mixture contained 100 g/ml heparin, 0.1 mM ATP, CTP, and GTP, and 1.5 Ci of [␣-32 P]UTP. Reactions were done at 37°C and stopped with 4 l of formamide dye mixture. 7 l 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 54 , 20 54 , and 46 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-l reactions) using 20 nM S. meliloti nifH early melted promoter probe (template strand 32 P end-labeled), 50 nM core RNAP, and 300 nM 54 in cleavage buffer (40 mM HEPES, pH 8.0, 10 mM MgCl 2 , 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 l of stop buffer (0.1 M thiourea and 100 g/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.

Core RNAP Lacking the ␤ Flap Forms an Unstable 54 Holoenzyme
Previously, we have demonstrated that deletion of the ␤ flap does not significantly affect the ability of mutant core RNAP to bind 70 (13). We tested the ability of ⌬flap E to bind 54 . For this purpose, a fixed amount of 32 P-labeled 54 ( 32 P-54 ) was combined with various amounts of ⌬flap E 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 32 P-54 was in the holoenzyme form (E 54 ) when the molar ratio of core RNAP to 54 reached 1:1. In contrast, a 4-fold excess of ⌬flap E was required to convert 32 P-54 to ⌬flap E 54 . Thus, it appears that ⌬flap E has a reduced ability to bind 54 , suggesting that the ␤ flap contributes to the 54 binding site.
To estimate the stability of the ⌬flap E 54 complex, we conducted experiments in which unlabeled 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 32 P-54 to capture free core RNAP arising from dissociation of the preformed holoenzyme complex. As shown in Fig. 2b, little formation of the 32 P-labeled RNAP holoenzyme was detected 1 min after the addition of 32 P-54 to the wild-type RNAP holoenzyme (lane 1), indicating that the wild-type E 54 complex had not yet dissociated. Strikingly, ϳ6-fold increased formation of 32 P-labeled RNAP holoenzyme complex was observed in the case of ⌬flap E 54 (Fig. 2b, lane 2), indicating that the mutant holoenzyme dissociated much faster than did the wild-type E 54 . The presence of promoter DNA did not markedly improve the stability of ⌬flap E 54 (data not shown). Overall, the data suggests that the ␤ flap contributes to the binding of 54 to RNAP core and the stability of the resulting RNAP holoenzyme. This observation is consistent with previous results in which a derivative of 54 harboring a FeBABE at residue Cys 198 within the major core RNAP binding surface of 54 (residues 120 -215; Fig. 1a) cleaved the RNAP ␤ subunit between residues 850 and 890 (9,20), close to or within the ␤ flap domain (residues 885-914).

The ␤ Flap Contributes to Heparin-stable Promoter Complex Formation by E 54
In E 70 , 70 region 4 (which recognizes the Ϫ35 promoter consensus element) interacts with the ␤ flap (2, 13). This interaction is functionally important and is required for correct positioning of the 70 region 4 to allow promoter complex formation on promoters of the Ϫ35/Ϫ10 class (2,13). To investigate the contribution of the ␤ flap domain to promoter binding by E 54 , we analyzed promoter complexes formed by ⌬flap E 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, ⌬flap E 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 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 E 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 E 54 promoter complex on the homoduplex promoter probe is a hallmark of either open or initiated E 54 promoter complexes. The results were quantified and are presented in Table I. Under nonactivating conditions ⌬flap E 54 bound the homoduplex probe with similar activity as E 54 , and the complexes were sensitive to heparin, as expected. Promoter complex formed by the wild-type E 54 became heparinstable under activating or initiating conditions, as expected. In contrast, neither activation nor initiation conditions led to the acquisition of heparin stability by ⌬flap E 54 on the homoduplex promoter (Table I). Because in the absence of heparin ⌬flap E 54 and E 54 bound the homoduplex probe equally well, we conclude that (i) the ␤ flap contributes to DNA opening and/or engagement of single-stranded DNA by E 54 to acquire heparin stability, or (ii) deletion of the ␤ flap compromises the ability of the mutant RNAP to respond to activation conditions. 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 Ϫ10 to Ϫ1 relative to the transcription start point and is believed to represent the conformation of promoter DNA in E 54 open complexes (21,22) (Fig. 1c). E 54 efficiently forms heparin-stable promoter complexes on the late-melted promoter probe only under activating conditions. Initial binding assays with ⌬flap E 54 in the absence of heparin showed that ⌬flap E 54 bound the late-melted probe with wild-type activity under all the conditions tested (Table I). Like E 54 , ⌬flap E 54 was unable to form heparin-stable complexes in the absence of activation (Table I). Activation (ϩPspF-(1-275) and ATP) increased the heparin stability of wild-type E 54 . In contrast, activated ⌬flap E 54 late melted promoter DNA complexes were heparin-sensitive (Table I). However, under activating conditions that permitted initiation (ϩPspF-(1-275) and GTP) a significant number of heparin-stable ⌬flap E 54 promoter complexes was detected (Table I), suggesting that (i) ⌬flap E 54 was able to respond to activation when the template strand was available as single-stranded DNA, and (ii) initiation of RNA synthesis stabilized ⌬flap E 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 E 54 , for example, those lacking the 54 regulatory Region I (⌬RI 54 ) (21) (Fig. 1a). To further assess the ability of ⌬flap E 54 to engage pre-melted promoter DNA in a heparinstable manner we conducted experiments with double mutant holoenzyme lacking the ␤ flap and 54 regulatory Region I. Initially, we tested whether ⌬RI 54 can form holoenzyme complexes with ⌬flap E. The results showed that a 1:8 ratio of ⌬flap E to ⌬RI 54 was needed to form stable ⌬flap E⅐⌬RI 54 complexes, consistent with the 54 binding defects of ⌬flap E (data not shown and Fig. 2). In the absence of heparin, ⌬flap E⅐⌬RI 54 bound the late melted promoter probe with an activity similar to that of E⌬⅐RI 54 (data not shown). Strikingly and in marked contrast to E⌬⅐RI 54 complexes, very little (Ͻ2%) of the ⌬flap E⅐⌬RI 54 late melted promoter complexes survived heparin challenge (Fig. 3, compare lane 2 with 5). However, conditions that permitted activator-independent initiation by E⌬⅐RI 54 (i.e. the presence of GTP) detectably stabilized ⌬flap E⅐⌬RI 54late melted promoter complexes (Fig. 3, compare lane 5 with 6). In contrast, the presence of noninitiating nucleotides, ATP or 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. dGTP, did not lead to heparin resistance of ⌬flap E⅐⌬RI 54 promoter complexes (data not shown). We note that increased heparin resistance of ⌬flap E⅐⌬RI 54 promoter complexes was not dependent on the presence of activator (Fig. 3, compare  lanes 6 and 7), suggesting that the deregulated properties of RI 54 have not been altered in the absence of the ␤ flap. Similar binding patterns were obtained with ⌬flap E in the context of other deregulated mutants of 54 , such as F318A (23), R336A (24), and Ala24 -26 54 (16), further confirming that initiation of RNA synthesis stabilizes late melted promoter complexes in the context of ⌬flap RNAP (data not shown).
Overall, several conclusions can be derived from the binding properties of ⌬flap E 54 on the late melted promoter probe. First, DNA interactions with melted DNA made by E 54 are destabilized in the absence of the ␤ flap. Second, ⌬flap E 54 closed complexes respond to activator and engage pre-melted DNA to form a heparin-stable complex, albeit less efficiently than the wild-type E 54 . This result suggests that the ␤ flap is not essential to the RNAP site that interacts with the activator. Third, the deletion of the ␤ flap does not deregulate E 54 , at least in the context of the late melted promoter probe complex.

DNase I Footprinting Reveals Altered Interactions of ⌬flap E 54 with the S. meliloti nifH Promoter
To further characterize the marked differences in promoter complex stability caused by deletion of the ␤ 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 E 54 and ⌬flap E 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 E 54 on the S. meliloti nifH homoduplex probe was typical of E 54 closed promoter complexes. Promoter DNA was protected from DNase I cleavage between positions Ϫ34 and Ϫ5 with respect to the transcription start site at ϩ1 (Fig. 4a (i), lane 3). The ⌬flap E 54 closed promoter complex footprint was similar to that of wildtype E 54 between positions Ϫ34 and Ϫ4 (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 (Ϫ34 to Ϫ4) 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 ⌬flap E 54 closed promoter complex or (ii) nonspecific binding of the ⌬flap E 54 . In promoter complexes formed with the wild-type E 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 ⌬flap E 54 (Fig. 4a (i), compare lanes  4 and 5). Strikingly, conditions that allowed initiation from ⌬flap E 54 promoter complexes resulted in increased DNase I cleavage between Ϫ34 and Ϫ5 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 ⌬flap E 54 formed an equal number of initiated promoter complexes as E 54 , suggesting that the increased cleavage of DNA between the Ϫ34 and Ϫ5 positions was not because of simple dissociation of promoter complexes (data not shown and Table  I). Overall, the DNase I footprinting data suggests that ⌬flap E 54 promoter complexes differ significantly from promoter complexes formed by E 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 ⌬flap E 54 closed complexes to activation   ) was used in all assays. b Indicates the % of DNA in complex with RNAP. c The bold values in parentheses indicate the % of initial promoter complex that survived a 5-min heparin challenge.

⌬flap E 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 ⌬flap E 54 and the wildtype E 54 by KMnO 4 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 ⌬flap E 54 . However, very little deregulated DNA melting was observed within the wild-type closed promoter complex. Activation of E 54 promoter complexes resulted in increased KMnO 4 reactivity of template strand thymines at positions Ϫ10 and Ϫ8 indicating promoter opening in response to activation (Fig. 4b, (ii)). In contrast, the KMnO 4 reactivity of the Ϫ10 and Ϫ8 thymines was greatly reduced within activated mutant promoter complexes. Under initiating conditions, a 2-fold further increase in KMnO 4 reactivity of thymine at po-sition Ϫ8 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 KMnO 4 reactivity was detected in ⌬flap E 54 -initiated promoter complexes. Overall, KMnO 4 probing data are consistent with DNase I footprinting results and strongly suggests that ⌬flap E 54 promoter complexes are defective for (i) DNA melting and/or (ii) activator responsiveness.

⌬flap E 54 Forms a Stable Complex with the 54 Activator PspF-(1-275)
To determine whether the absence of the ␤ flap affects the ability of E 54 to form a stable binary complex with 54 activator PspF-(1-275), we conducted binding assays in the presence of ADP-AlF x , a nonhydrolysable analogue of ATP that allows stable activator-E 54 complexes to form (18). E 54 or ⌬flap E 54 were combined with PspF-  in the presence of ADP-AlF x and protein complexes were analyzed by native PAGE. Fig. 5 shows that both holoenzymes formed similar amounts of stable, ADP-AlF x -dependent complexes with PspF-  Thus, it appears that the greatly reduced DNA melting in ⌬flap E 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 ␤ flap is not part of the putative ␤ subunit activator interaction site previously detected by chemical crosslinking (25).

⌬flap E 54 Is Defective for Activator-dependent in Vitro Transcription from Supercoiled Templates
To assess the consequence of 54 -binding and promoter DNAinteraction defects of ⌬flap E 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, [␣-32 P]UTP, and the remaining nucleotides, ⌬flap E 54 was only about 15% as active as the wild-type E 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 ⌬flap E 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, ⌬flap E 54 showed less than 5% of the wild-type E 54 activity, indicating a destabilization of complexes under initiating conditions. This result is striking as the presence of GTP-stabilized ⌬flap E 54 and ⌬flap E⌬R1 54 complexes on late melted promoter (Table I and Fig. 3, respectively). However, the result is consistent with the DNase I and KMnO 4 footprinting data, where initiation resulted in an altered DNase I footprint for ⌬flap E 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 ⌬flap E 54 . Overall, the in vitro transcription data from supercoiled templates show that ⌬flap E 54 is transcriptionally active, but to a lesser degree than E 54 . A likely explanation for this could be the weakened 54 and promoter DNA interactions made by E 54 in the absence of the ␤ flap.

⌬flap E 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 ⌬flap E 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, [␣-32 P]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 ⌬flap E 54 was 40% as active as the wild-type E 54 (Fig. 6c,  compare lanes 3 and 4). This run-off transcript was of the expected size (ϳ28 bp) and its production was 54 and activatordependent (Fig. 6c, lanes 1, 2 and 5, 6, respectively). In the presence of GTP and PspF-(1-275), transcription by the ⌬flap E 54 was poor, resulting in ϳ12% of wild-type activity. ⌬flap E 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 E 54 associated with the ⌬flap mutation can be overcome by using pre-melted promoter templates. Overall, the single-round and run-off transcription data, together with the KMnO 4 probing of mutant promoter complexes, strongly implies that the ⌬flap E 54 is defective for stable DNA melting in response to activation.  For a and b, the percentage of transcripts produced with respect to wild-type E 54 is given. c, activator-dependent in vitro transcription activity of E 54 and ⌬flap E 54 from the S. meliloti nifH late melted promoter probe. Lane 9 contains molecular weight markers and the activity of the ⌬flap E 54 is given with respect to wild-type E 54 activity.  54 and R336A 54 in the context of the ⌬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.
[␣-32 P]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 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 ⌬flap E and either of the deregulated 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 ⌬flap E 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 ⌬flap E 54 were defective for promoter DNA binding. However, DNase I footprints of closed complexes formed with deregulated forms of ⌬flap E 54 indicated that this was not the case (data not shown).
Overall, it appears that removal of the ␤ flap while only partially affecting the ability of wild-type 54 RNAP holoenzyme to transcribe from supercoiled templates, completely abolishes transcription by RNAP holoenzymes reconstituted with deregulated forms of 54 . This suggests that the ␤ flap is required for unstable open promoter complex formation and/or that the ␤ flap is needed for transcription initiation from the unstable promoter complexes.

⌬flap E 54 Makes Altered DNA Interactions within Closed Promoter Complexes
The apparently contradicting properties exhibited by deregulated forms of ⌬flap E 54 in the late melted promoter probe binding experiments and transcription assays prompted us to investigate DNA interactions made by ⌬flap E 54 early during open promoter complex formation. The transcriptionally silent state of the E 54 closed promoter complexes strictly depends on the integrity of promoter sequences immediately downstream of the Ϫ12 promoter consensus element (the GC-element; Fig.  1c). In wild-type E 54 closed promoter complexes, the base pair immediately downstream of the GC element shows increased reactivity toward KMnO 4 , 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 E 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 E 54 (28). To investigate early DNA melting within closed promoter complexes formed by ⌬flap E 54 , ortho-CuOP footprinting was performed. As expected, DNA cleavage by ortho-CuOP was seen at position Ϫ12 (immediately downstream the GC-element; Fig. 1c) in closed complexes formed with the wild-type E 54 on the S. meliloti nifH homoduplex promoter probe (Fig. 8a, lane 3). Activation of the wild-type closed complexes resulted in ϳ5-fold reduction of the Ϫ12 signal, indicating open complex formation (Fig. 8a,  compare lanes 3 and 5). In contrast, cleavage at Ϫ12 was absent in closed complexes formed by ⌬flap E 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 E 54 protected the mismatched DNA region from ortho-CuOP cleavage, indicating the binding of E 54 to the promoter probe (Fig. 8b, compare lanes 1 and 3). A clear protection of the hypersensitive site at Ϫ12 was also seen in reactions containing ⌬flap E 54 indicating complex formation by ⌬flap E 54 on the early melted promoter probe (Fig. 8b, lane 4). DNase I footprinting experiments also showed protection between DNA positions Ϫ34 to Ϫ5 in the presence of both E 54 and ⌬flap E 54 (Fig. 8c, compare lanes 3 and 4), thus confirming efficient complex formation on early melted promoter probe by both enzymes.
To gain further insights into ⌬flap E 54 interaction with early melted DNA, we tested heparin stability of nifH promoter early melted complexes. As summarized in Table I, ⌬flap E 54 bound the early melted promoter probe as efficiently as wild-type E 54 under all conditions tested. As reported previously (29,30), and in contrast to the situation on homoduplex promoter probes, conditions that permitted activation (ϩPspF-(1-275) and ATP) or initiation (ϩPspF-(1-275) and GTP) did not markedly increase the heparin stability of wild-type E 54 complexes on the early melted promoter probe (Table I). This property of wildtype E 54 complexes is attributed to tight binding of 54 to the heteroduplex region of the early melted promoter probe (15,29). Significantly, the ⌬flap E 54 -early melted promoter probe complex was unstable and did not survive heparin challenge under any of the conditions tested (Table I), suggesting that E 54 -early melted DNA interactions are altered in the absence of the ␤ 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 in the presence of heparin. However, band shift analysis of holoenzymes on native gels similar to the ones shown in Fig. 2 showed that ⌬flap E 54 was as sensitive to disruption by heparin as the wild-type E 54 (data not shown). Thus, the failure of the ⌬flap E 54 to form heparin-stable complexes on the early melted promoter probe seems to be because of a failure of the ⌬flap core RNAP to bring about a DNA-dependent change(s) in the core RNAP-54 interaction that results in the formation of heparin-stable promoter complexes. In other words, we suggest that ⌬flap E 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 E 54 Closed Complexes Is Dependent on the ␤ Flap
Regulated transcription by E 54 relies on the integrity of 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, 54 Region I localizes proximal to the ␤ and ␤Ј subunit residues that contribute to the formation of the RNAP active center (9). Region I is directly contacted by activators of E 54 transcription (18). We have termed the 54 Region I-DNA-core RNAP arrangement within closed E 54 promoter complexes the regulatory center (19). Experiments presented above suggest that some of the properties of ⌬flap E 54 could be accounted for by defects in the organization of the regulatory center. To investigate whether the ␤ flap contributes to proper positioning of 54 Region I and hence to the organization of the regulatory center, we conducted localized hydroxyl radical cleavage experiments using FeBABE derivatives of 54 . RNAP holoenzymes were reconstituted with the wild-type and mutant RNAP core enzymes and 54 containing FeBABE conjugated to Cys 20 and Cys 46 ( 20 * 54 and 46 * 54 ) 3 in Region I 4 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 * 54 and 46 * 54 formed promoter complexes on the early melted promoter probe as efficiently as holoenzymes reconstituted with unmodified 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 54 ( Cys-free 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 Ϫ11 and Ϫ16 was seen within promoter complexes formed by E 20 * 54 (Fig. 9, lanes 3 and 4), suggesting that 54 Region I residue 20 is proximal to the regulatory center. Strikingly, no DNA cleavage was detected in complexes formed by ⌬flap E 20 * 54 (Fig. 9, compare lanes 3 and  FIG. 9. FeBABE footprints of S. meliloti nifH early melted promoter probe. FeBABE cleavage profiles of S. meliloti nifH template strand by 20 * 54 , 46 * 54 , and Cys-free 54 in the context of the wild-type and ⌬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. 4 with 9 and 10), suggesting that in the absence of the ␤ flap, residue 20 in 54 Region I is unable to occupy its correct position in the regulatory center. In E 46 * 54 complexes, cleavages in the template strand of promoter DNA between positions Ϫ6 and Ϫ2 were obtained (Fig. 9, compare lanes 5 and 6), indicating that residue 46 in 54 Region I is localized outside of the regulatory center. Interestingly, ⌬flap E 46 * 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 54 Region I residues that do not contribute to the regulatory center is not affected by the ⌬flap mutation, at least in the context of this assay. Thus, this suggests that the ␤ flap directly and selectively contributes to the organization of the E 54 regulatory center. DISCUSSION Transcription from enhancer-dependent promoters relies upon the RNAP containing the 54 factor. Unlike the enhancerindependent promoters recognized by the 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 AAAϩ protein family (4,35). The contribution of the RNAP mobile structural modules (␤ flap, ␤Ј clamp, ␤ downstream and upstream lobes) to enhancer-dependent and enhancer-independent transcription by E 54 and E 70 , respectively, is not well understood at a molecular level. By using a mutant RNAP harboring a deletion of the ␤ flap (⌬885-914; ⌬flap), we have investigated the contribution of this RNAP structural module to E 54 functioning. The data establish that the ␤ flap domain has multiple roles in enhancer-dependent transcription. Strikingly, the ␤ flap domain appears to have different functionalities in enhancer-dependent and enhancer-independent transcription.
54 Binding-Even though the enhancer-dependent 54 and the enhancer-independent 70 bind the common RNAP with similar affinities and occupy similar positions within the RNAP holoenzyme (7,9,36), the ⌬flap mutation markedly reduces the activity of the RNAP to bind 54 and the stability of the resulting holoenzyme. In contrast, 70 binding and the stability of E 70 were not greatly affected by the ⌬flap mutation (13). Holoenzyme formation is accompanied by large-scale conformational changes of all the structural modules of the ␤ and ␤Ј subunits that serve to orientate and position the factor for promoter-specific transcription initiation. Upon binding 70 , the ␤ flap re-positions region 4 of 70 to facilitate recognition of the Ϫ35 promoter element (2,3,13). The contribution of the ␤ flap to E 54 formation appears to be 2-fold. First, the instability of the ⌬flap E 54 complexes suggests that the ␤ flap contributes to the anchoring of 54 to the core RNAP. Second, differences in the DNA cleavage patterns by FeBABE-modified 54 in the context of the wild-type and ⌬flap RNAP imply a role for the ␤ flap in the proper positioning of 54 domains within the holoenzyme that is important for E 54 functioning.
Promoter Complex Formation-Promoter complex formation by E 70 induces major conformational changes in two of the mobile modules of the RNAP, notably also including the ␤ flap domain (3). Consequently, E 70 with the ⌬flap mutation is unable to efficiently bind and utilize the Ϫ35/Ϫ10 class of bacterial promoters (13). In the context of the E 54 , the ⌬flap mutation has little effect on initial promoter complex formation as judged by gel shift and DNase I footprinting assays. In contrast to E 54 , ⌬flap E 54 promoter complexes formed under activating conditions were sensitive to heparin and KMnO 4 probing showed greatly reduced DNA opening within such mutant promoter complexes. Consequently, in vitro transcription activity of the ⌬flap E 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 ⌬flap E 54 . In contrast, DNA opening was fully normal in promoter complexes formed with the ⌬flap E 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 ⌬flap E 54 proceed via an altered pathway that neither results in efficient DNA melting nor acquisition of heparin stability. The limitation of the ⌬flap E 54 to only function with the wild-type 54 for transcription, but not with deregulated 54 mutants that transcribe via unstable and heparin-sensitive open complexes, supports this view. Thus, we propose that the ␤ flap contributes to the formation of an intermediate, heparin-sensitive promoter complex state or states en route to heparin stable E 54 open complex formation.
Acquisition of Heparin Stability-On the late melted promoter probe, activated E 54 promoter complexes are resistant to heparin, suggesting that the E 54 must undergo conformational changes in response to activation to acquire stability (21,22). The ⌬flap E 54 does not efficiently form heparin-stable complexes on the late-melted promoter under activating conditions, suggesting that, in the absence of the ␤ flap, the E 54 does not undergo the same set of conformational changes that leads to heparin stability. Furthermore, unlike the wild-type E 54 , the ⌬flap E 54 also forms heparin-sensitive complexes on the early melted promoter probe under nonactivating conditions. Thus, we propose that the ␤ flap makes a significant contribution to the acquisition of heparin stability by the E 54 . Previously, we have reported that either the absence of 54 Region I or mutations affecting the ␤ subunit downstream lobe functionality reduces the ability of the E 54 to form heparin-stable promoter complexes (10,29). We envisage a functional co-operation between Region I of 54 and two mobile structural modules, the ␤ flap and the ␤ downstream lobe, which leads to heparin stable-promoter complex formation during enhancer-dependent transcription.
Organization of the E 54 Regulatory Center-In enhancerdependent closed complexes formed by the E 54 , Region I of 54 , and the catalytic center of the RNAP co-localize over the Ϫ12 position (GC-element) of the promoter where DNA melting originates. This nucleoprotein complex is defined as the E 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 ⌬flap RNAP clearly indicates that residues in Region I of 54 that contribute to the regulatory center are not proximal to the regulatory center in the absence of the ␤ flap domain. This suggests that the ␤ 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 ⌬flap E 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 conforma-5 K. Severinov and K. Kuznedelov, unpublished observation. tional 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, factors make extensive interactions with the structural mobile modules (␤Ј clamp, ␤ flap, and ␤ downstream and upstream lobes) of the bacterial RNAP during the process of transcription initiation (1,2,40). Unlike the ␤ downstream lobe module, which is commonly used by E 54 and E 70 during open complex formation (8,10), the ␤ flap module makes markedly different contributions to enhancerdependent and -independent transcription by E 54 and E 70 , respectively. For E 70 , the ␤ flap plays a crucial role in the proper positioning of 70 region 4 within the holoenzyme for promoter-specific transcription initiation (13). Thus, it appears that for enhancer-independent transcription by E 70 , the ␤ flap contributes to RNAP-promoter DNA interactions at the start site distal promoter element. For E 54 , the ␤ flap has multiple roles in modulating E 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 54 Region I or other parts of 54 that interact with the ␤ flap en route to enhancer-dependent transcription initiation. However, the data presented here highlights the fact that the extensive interface between factor and RNAP is crucial for the co-ordinated reconfiguration of both partners for efficient transcription initiation.