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J. Biol. Chem., Vol. 278, Issue 32, 29728-29743, August 8, 2003

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

Patricia C. Burrows, Konstantin Severinov , Akira Ishihama , Martin Buck ¶ and Siva R. Wigneshweraraj ||

From the Department of Biological Sciences, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom, Waksman Institute and Department of Genetics, Rutgers, The State University, Piscataway, New Jersey 08904, and Nippon Institute for Biological Science, Shin-machi 9-2221, Ome, Tokyo 198-0024, Japan

Received for publication, April 7, 2003 , and in revised form, May 12, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Transcription of DNA is a fundamental process needed for regulation of cellular adaptation and differentiation and is carried out by DNA-dependent RNA polymerases (RNAPs).¹ Multisubunit RNAPs are evolutionarily and structurally conserved enzymes. In bacteria, promoter-specific transcription initiation requires the binding of a -subunit to the catalytically proficient core RNAP (subunit composition ₂') to form the holoenzyme (-RNAP). Bacterial -factors are categorized into two types (1, 2). The ⁷⁰-type, named after the prototypical housekeeping -factor of Escherichia coli, ⁷⁰, includes most bacterial -factors, and ⁵⁴ proteins are clearly different (3, 4). Although both types bind the same core RNAP, their holoenzymes differ markedly in their control (3, 5).

The ⁵⁴-RNAP recognizes promoters with conserved regions at positions –24 (the GG-region) and –12 (the GC-region) with respect to the transcription start site (Fig. 1a) (6). Open complex formation requires a class of activator proteins that bind to enhancer-like sequences and interact with the closed promoter complex through a DNA looping event (7–9). These activators are ATPases that belong to the AAA (ATPases associated with various cellular activities) protein family. ATP hydrolysis brings about conformational changes in the ⁵⁴-RNAP-closed complex that lead to open complex formation (10–12). In contrast, ⁷⁰-type RNAPs use promoters characterized by conserved DNA sequences located at –10 and –35, where the closed to open complex transition occurs spontaneously and independently of an ATP energy source (13).

View larger version (24K): [in this window] [in a new window]   FIG. 1. a, nucleotide sequence of the S. meliloti nifH promoter. Boxed are consensus hexanucleotide sequences centered at –24 (5'-CTGGCA-3') and –12 (5'-TTTGCA-3') positions with respect to the transcription start site at +1 (indicated by the thin arrow). The GG and GC dinucleotides (boldface type) characterize the 54 binding sites. The two thick arrows indicate positions on the nontemplate strand (A and C) that were changed to C and A to create the heteroduplex early melted probe. b, domain organization of the K. pneumoniae 54. The domains and residues involved in core RNAP, activator, and DNA interactions are indicated. In Region III, boxed are residues with potential roles in DNA interactions: the putative helix-turn-helix (HTH) motif (gray box) (19, 55), a region that has been shown to cross-link to DNA (black box) (56), and the RpoN box motif (white box) (16, 20). The regions that were targeted for introducing a cysteine residue for FeBABE conjugation (residues 39–48 and 453–476) are expanded and aligned with corresponding regions in 54 from E. coli (Ec), Salmonella typhimurium (St), S. meliloti (Sm), Pseudomonas aeruginosa (Pa), Planctomyces limnophilus (Pl), Bacillus subtilis (Bs), and Aquifiex aeolicus (Ae). Highly conserved amino acid residues are in boldface type. The asterisk indicates the amino acid residues (Leu46, Arg455, Lys460, Glu463, and Lys474) that were targeted for cysteine substitutions and FeBABE conjugation.

The –12/–24 regions of ⁵⁴-dependent promoters might be considered as functional analogues of the –10/–35 regions of ⁷⁰-dependent promoters. Genetic, biochemical, and structural data show that the –10 and –35 regions of the ⁷⁰-dependent promoters are recognized by ⁷⁰ conserved regions 2.4 and 4.2, respectively (14, 15). In contrast, the precise ⁵⁴ sequences involved in recognizing the –12 and –24 regions of ⁵⁴-dependent promoters are not well known.

The functional domain organization of ⁵⁴ is complex and clearly different from that of ⁷⁰ (3, 4). Extensive deletion and mutational analyses of ⁵⁴ have allowed functions to be assigned to different regions of the protein (Fig. 1b). Recognition of the –12 GC-region, where DNA melting originates, involves ⁵⁴ Region I and Region III sequences (16–19). The RpoN box, a signature sequence found in ⁵⁴, has been proposed to interact with the –24 GG-region, but no physical or genetic suppression evidence has been obtained to directly establish this view (16, 20).

E. coli ⁷⁰ undergoes core RNAP-mediated conformational changes that allow the recognition of the consensus –10 and –35 promoter elements (21–24). Significant conformational changes may also occur during ⁵⁴-RNAP promoter complex formation. Unlike ⁷⁰, ⁵⁴ can bind to promoter DNA in the absence of core RNAP (25). However, ⁵⁴-RNAP footprints the –12 GC-region differently from ⁵⁴, suggesting that core RNAP changes ⁵⁴-promoter interactions in some way (26). The protein-DNA arrangement at the –12-position, termed the regulatory center (17, 27), undergoes conformational changes during activation.

The information about ⁵⁴ regions involved in interactions with the consensus –24 promoter region and the dynamics of these interactions during open complex formation and transcription initiation is sparse. To address this issue, we converted ⁵⁴ to a proximity-based cleavage reagent through the conjugation of a reactive Fe²⁺ chelate using [(p-bromoacetamidobenzyl)-EDTA Fe] (FeBABE) (28) and now provide the first physical evidence in favor of an interaction between the RpoN box and the consensus –24 promoter region and ⁵⁴ Region I and the consensus –12 promoter region (Fig. 1, a and b). Overall, the changes in the spatial relationships during transcription initiation between ⁵⁴ and promoter DNA provide insights into how conformational changes mediate the progression of transcription events at ⁵⁴-dependent promoters. The approaches described within this report can be applied to study static and dynamic nucleoprotein complexes for which little or no structural data are available.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Proteins and Promoter DNA Probes—Klebsiella pneumoniae cysteine-free ⁵⁴ and single-cysteine variants (Table I) thereof were constructed and purified as amino-terminal His₆-tagged fusion proteins essentially as described in Ref. 29. The catalytic domain (residues 1–275) of the ⁵⁴-dependent activator phage shock protein F (PspF_1–275) was purified as described in Ref. 30. E. coli core RNAP containing the -subunit flap deletion (885–914) was purified as described in Ref. 31. Wild-type E. coli core RNAP was purchased from Epicentre Technologies. Three types of Sinorhizobium meliloti nifH promoter probes were used for the FeBABE cleavage experiments. The homoduplex and early melted probes were prepared as described in Ref. 32. Supercoiled plasmid pMKC28 containing the S. meliloti nifH promoter in pTE103 (18) was prepared from E. coli strain MC1061 using the Qiagen Maxi Prep plasmid purification kit according to the manufacturer's instructions.

View this table: [in this window] [in a new window]   TABLE I In vitro transcription properties of single-cysteine 54 Variants in the absence and presence of FeBABE

Conjugation with FeBABE—The single-cysteine variants of ⁵⁴ were conjugated with FeBABE as previously described in Ref. 29. The conjugation reactions were conducted in MOPS buffer (10 mM MOPS (pH 8.0), 200 mM NaCl, 2 mM EDTA, 5% glycerol) at 37 °C using a 20-fold molar excess of FeBABE over ⁵⁴. Where necessary, MOPS buffer with 6 M urea was used for conjugation under denaturing conditions. Conjugation efficiencies were measured fluorometrically using N-[4-[7-(diethylamino)-4-methylcoumarin-3-yl]malemide (CPM) as previously described in Refs. 29 and 33.

In Vitro Transcription Assays—Reactions (10 µl final volume) were conducted using 200 nM core RNAP reconstituted with a 1:3 molar ratio of core RNAP to ⁵⁴ (FeBABE-conjugated and -unconjugated) and 10 nM pMKC28. Plasmid pMKC28 contains 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 of a discrete transcript of 470 nucleotides. Assays were performed 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) essentially as described in Ref. 29. For open complex formation, 5 µM PspF_1–275 and 4 mM ATP were used. The elongation mixture contained 100 µg/ml heparin; 0.1 mM ATP, CTP, and GTP; and 0.05 mM UTP (0.25 µCi of [-³²P]UTP (800 Ci/mmol)). Reactions were conducted 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 to measure the transcriptional activities (percentage) relative to wild-type ⁵⁴. Since the conjugation yields were not 100%, the total activity a_tot (in arbitrary units) of ⁵⁴ after conjugation is the weighted sum of the activities contributed by both (unconjugated and conjugated) ⁵⁴ species: a_tot = (1 – f_uc)a_c + f_uca_uc, where f_uc represents the fraction unconjugated, a_uc (in arbitrary units) is the activity of the unconjugated ⁵⁴, and a_c is the activity of the conjugated ⁵⁴ (in arbitrary units). The above equation was used to calculate the percentage of transcription activity relative to the wild-type ⁵⁴ of the single-cysteine ⁵⁴ following FeBABE conjugation.

Native Gel Mobility Shift Assays—These were conducted essentially as described in Ref. 12 in STA buffer. Binding reactions (10 µl) were conducted using 1 µM FeBABE-conjugated ⁵⁴ and 20 nM early melted probe. Free DNA was separated from ⁵⁴-bound DNA on a native 4.5% polyacrylamide gel run in 25 mM Tris, 200 mM glycine buffer at room temperature.

DNA Cleavage Assays—DNA cleavage assays were conducted at 37 °C in cleavage buffer (40 mM HEPES (pH 8.0), 10 mM MgCl₂, 5% (v/v) glycerol, 0.1 M KCl, and 0.1 mM EDTA). Where indicated, 1 µM FeBABE-conjugated ⁵⁴, 200 nM ⁵⁴-RNAP, 5 µM PspF, 4 mM NTPs (ATP or GTP), 100 µg/ml heparin, and 10 nM promoter template (homoduplex probe, early melted probe, or pMKC28) were used. In assays using the flap core RNAP, ⁵⁴-RNAP was reconstituted with a 1:4 molar ratio of flap core RNAP to ⁵⁴. Where indicated, ascorbate (pH 7.0) and hydrogen peroxide were used at final concentrations of 2 and 1 mM, respectively.

Linear Template Cleavage—Binding assays were conducted using the homoduplex or early melted promoter probes reconstituted with either the nontemplate or template strand end-labeled with -³²P. Following incubation for 5 min, cleavage was initiated by the sequential addition of ascorbate and hydrogen peroxide. The reactions were allowed to proceed for 10 min before quenching with 30 µl of stop buffer (0.1 M thiourea and 100 µg/ml sonicated salmon sperm DNA). The stopped reactions were phenol/chloroform-extracted and precipitated with ethanol. Recoveries of DNA were determined by dry Cerenkov counting, and equal numbers of counts were loaded onto 10% denaturing gels. Dried gels were visualized and analyzed using a Phosphor-Imager. The cleavage sites were determined by using -³²P end-labeled fragments of the S. meliloti nifH promoter DNA.

Supercoiled Template Cleavage—Binding and cleavage reactions were conducted as described above. For open complex formation, ATP and a truncated form of the ⁵⁴-dependent activator (PspF_1–275) that lacks the DNA binding domain and activates transcription from solution (30) were added for 10 min. For initiated complex formation, PspF_1–275 and GTP were added for 10 min, which allows the synthesis of an RNA trimer from the S. meliloti nifH promoter (18). Where indicated, the heparin challenge was conducted for 2 min. Following cleavage, the reactions were stopped by purifying the cleaved DNA using QIAquick spin columns (Qiagen) according to the manufacturer's instructions. The eluate containing the cleaved DNA was dried using a speed-vacuum drier and resuspended in 10 µl of 1x Deep Vent (exo^–) DNA polymerase buffer (New England Biolabs). The sites of FeBABE-mediated cleavage on both promoter DNA strands were identified by primer extension PCR using -³²P-end-labeled primers pMKC-F (5'-gacactgtccgtagcccttgtcggc-3'; for the nontemplate strand) and pMKC-R2 (5'-gatggcagctctgcgtcagatcgcg-3'; for the template strand). The primer extension PCRs (final reaction volume of 6.2 µl) were conducted in Deep Vent (exo^–) DNA polymerase buffer (New England Biolabs) using 1 µl of cleaved template DNA (from above), 0.2 µl of 3% Triton X-100, 0.3 µl of 2.5 mM dNTP mix, 0.5 µl of 1 µM -³²P-endlabeled primer, and 0.3 µl of Deep Vent DNA polymerase (2000 units/µl). Following extension by PCR (30-s denaturing at 95 °C, 15-s annealing at 65 °C, and 40-s extension at 72 °C over 10 cycles), the reactions were stopped with 4 µl of formamide dye and loaded directly onto a 10% denaturing gel. The dried gel was visualized and analyzed as described above. The cleavage sites were determined by sequencing pMKC28 using the chain termination method either with -³²P-end-labeled primer pMKC-F or pMKC-R2 using the T7 Sequenase Quick-Denature plasmid sequencing kit (Amersham Biosciences).

Elongation Complex Formation—Elongation complexes where more than 3 nucleotides of RNA was synthesized were formed as outlined in Fig. 7. Briefly, ⁵⁴ Cys⁴⁶³-RNAP was incubated in cleavage buffer for 10 min at 37 °C with pMKC28 in the presence of 4 mM GTP, ATP, and CTP and 4 µM PspF_1–275 to allow synthesis of more than 3 and probably 9 nucleotides of RNA (5'-GGGCGCGCA-3') from the S. meliloti nifH promoter. FeBABE-mediated DNA cleavage within the stalled elongation complex was conducted and analyzed as described above. For transcription from the stalled elongation complex, 0.05 mM UTP (0.25 µCi of [-³²P]UTP (800 Ci/mmol) was added to the reaction and incubated for a further 10 min. The transcription reactions were stopped and analyzed as described above.

View larger version (24K): [in this window] [in a new window]   FIG. 7. The spatial relationship between 54 and the –24 promoter region changes during early elongation. The experimental strategy is outlined at the top. Early elongation complexes (54 Cys463-RNAPEC) were formed with 54-RNAP reconstituted with 1:1 molar ratio (400 nM) of FeBABE-conjugated 54 Cys463 and core RNAP. Cold nucleotides were used at 4 mM final concentration, and PspF1–275 was at 4 µM. Following challenge with heparin (100 µg/ml) and the addition of ascorbate and hydrogen peroxide, the reaction was split in two. a, nontemplate strand cleavage within promoter complexes formed on pMKC28 in the presence (lanes 1 and 2) and absence (lanes 3 and 4) of core RNAP. The dotted line in lane 2 indicates the "missing cleavage sites" at the –24 position within 54 Cys463-RNAPEC. b, transcription from the Cys463-RNAPEC promoter complex after adding [-32P]UTP. c, transcription from heparin-resistant initiated FeBABE-conjugated Cys463-RNAP promoter complexes (as in Fig. 6a, lanes 8 and 16) after adding [-32P]UTP. In a–c, the plus and minus signs indicate the presence and absence of ascorbate and hydrogen peroxide in the reactions, respectively.

View larger version (36K): [in this window] [in a new window]   FIG. 6. 54-RNAP-DNA interactions with the consensus –24 promoter region of 54-dependent promoter remain unchanged during open complex formation and transcription initiation. a, template and nontemplate strand cleavage by FeBABE-conjugated 54 Cys463-RNAP within the closed (lanes 1, 2, 9, and 10), open (lanes 3, 4, 7, 11, 12, and 15), and initiated (lanes 5, 6, 8, 13, 14, and 16) promoter complexes formed on pMKC28. b, PhosphorImager traces (shown here for the template strand) of closed, open, and initiated promoter complexes (black lines) formed by the Cys463-RNAP on pMKC28 under the cleavage conditions shown in a, lanes 10, 15, and 16, respectively, but in the absence of ascorbate and hydrogen peroxide. The DNA sequence (from position –34 to –5) that becomes footprinted by 54-RNAP is shown, and the location of the –12 and –24 GC- and GG-regions, respectively, are indicated. The DNA sequence that becomes protected only in open and initiated promoter complexes is shown by the dotted line. The free DNA that has been subjected to DNase I cleavage is shown in gray. c, template and nontemplate strand cleavage by 54 Cys46-RNAP within the closed (lanes 1, 2, 9, and 10), open (lanes 3, 4, 7, 11, 12, and 15), and initiated (lanes 5, 6, 8, 13, 14, and 16) promoter complexes formed on pMKC28. d, PhosphorImager traces (shown here for the template strand and annotated as in b) of closed, open, and initiated promoter complexes formed by 54 Cys46-RNAP on pMKC28 under the cleavage conditions shown in c, lanes 10, 15, and 16, respectively, but in the absence of ascorbate and hydrogen peroxide. In a and c, lanes marked A, C, G, and T contain chain termination DNA sequencing reactions conducted with pMKC28 and the nucleotide analogues ddATP, ddCTP, ddGTP, and ddTTP, respectively. The relevant DNA sequence is shown with the consensus GG- and GC-regions marked in boldface type. The black dots indicate the positions where FeBABE-mediated cleavage occurs. In a, the white dot indicates weak cleavage seen at positions –26 only in the presence of heparin. e, nontemplate strand DNA cleavage by 54-RNAP reconstituted with 54 variant harboring FeBABE at positions Leu46 and Glu463 demonstrating that the relationship between 54 Region I residue Leu46 and the consensus –12 promoter region has changed in open (lane 5) promoter complexes formed on pMKC28, whereas the relationship between the 54 RpoN box residue Glu463 and the consensus –24 promoter region remains unchanged. The dotted line in lane 5 indicates the absence of DNA cleavage at the –12 position. The figure is annotated as in a and c.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Creation of Functional ⁵⁴-FeBABE Derivatives
FeBABE is a bifunctional chelating agent that conjugates to the sulfhydryl group of cysteine residues. It can be introduced at single positions along a polypeptide chain by designing variants of the protein containing a unique accessible cysteine. After the addition of ascorbate and hydrogen peroxide, hydroxyl radicals are generated at the iron ion attached to the BABE, which cleave DNA and polypeptide chains in its proximity (12 Å plus the 3 Å diffusion distance of the hydroxyl radicals) (28). Previously, we constructed a functional K. pneumoniae rpoN (⁵⁴) gene in which two naturally occurring cysteine codons were replaced by alanine codons (29). We have introduced unique cysteines into several positions of the altered K. pneumoniae rpoN. Sites were chosen based on data that indicated that these positions would result in neither complete inactivation nor deregulation of ⁵⁴ (16, 20, 34).

Unique cysteines were introduced at a single position in Region I (Leu⁴⁶) and at three positions in the RpoN box (Arg⁴⁵⁵, Lys⁴⁶⁰, and Glu⁴⁶³) (Fig. 1b). A cysteine residue was also introduced at Lys⁴⁷⁴, the most C-terminal positively charged residue in ⁵⁴ (Fig. 1b), to orient the C terminus with respect to the RpoN box residues and the –24 promoter region. The resulting single-cysteine ⁵⁴ derivatives (Cys⁴⁶, Cys⁴⁵⁵, Cys⁴⁶⁰, Cys⁴⁶³, and Cys⁴⁷⁴) were assayed for activator-dependent in vitro transcription from the supercoiled S. meliloti nifH promoter (pMKC28). Assays were conducted with subsaturating amounts of ⁵⁴-RNAP to allow quantitative comparisons of ⁵⁴-RNAPs reconstituted with the mutant ⁵⁴ variants. ⁵⁴-RNAPs reconstituted with each single-cysteine ⁵⁴ subunit exhibited >50% activity compared with the wild-type ⁵⁴-RNAP (Table I). Replacement of all endogenous cysteines with alanine residues and the introduction of cysteine residues at selected positions clearly had no gross negative effect on the activity of ⁵⁴.

Each single-cysteine ⁵⁴ variant as well as the control cysteine-free protein Cys(–) ⁵⁴ were derivatized with the FeBABE reagent under native conditions, and the conjugation yield was determined (33). The conjugation yield for single-cysteine ⁵⁴ variants Cys⁴⁶, Cys⁴⁵⁵, and Cys⁴⁷⁴ was estimated to be >60% (Table I). The single-cysteine ⁵⁴ variants Cys⁴⁶⁰ and Cys⁴⁶³ proved difficult to conjugate under native conditions (conjugation yields <20%; data not shown). Since the hydrophobic reagent CPM reacted with high yield at cysteines 460 and 463 and the hydrophilic reagent FeBABE did not (data not shown), we suggest that residues 460 and 463 are probably slightly buried or located in a hydrophobic region in the folded state of ⁵⁴. When FeBABE conjugation reaction with Cys⁴⁶⁰ and Cys⁴⁶³ was performed under denaturing conditions, the renatured Cys⁴⁶⁰ and Cys^{463 54} variants exhibited conjugation yields of 63 and 95%, respectively (Table I). The Cys(–) ⁵⁴ variant did not react with CPM and FeBABE, clearly implying that under the conditions used, FeBABE conjugation occurred only at the free sulfhydryl group of cysteine residues (Table I).

To determine the effects of FeBABE conjugation on -RNAP formation and transcription, we performed in vitro activator-dependent single-round transcription assays. After correcting for the presence of unconjugated ⁵⁴, the ⁵⁴-RNAPs reconstituted with FeBABE-conjugated Cys⁴⁵⁵, Cys⁴⁶³, and Cys^{474 54} variants showed >55% transcription activity compared with the wild-type ⁵⁴-RNAP (Table I). In contrast, ⁵⁴-RNAPs reconstituted with FeBABE-conjugated Cys⁴⁶ and Cys^{460 54} variants showed about 40% of transcription activity (Table I), suggesting that the presence of the 490-Da FeBABE molecule at positions 46 and 460 somehow modestly interferes with one or more events of the transcription reaction. Nevertheless, since all FeBABE-modified ⁵⁴-RNAPs were significantly active for promoter-complex formation and transcription initiation (data not shown; Table I), we conclude that the cleavage pattern by hydroxyl radicals should be a reasonable marker of the distance between the tethered residue and the DNA bases at which the cleavage occurs.

Promoter DNA Cleavage by FeBABE-conjugated ⁵⁴ and ⁵⁴-RNAP
We used a heteroduplex variant of the S. meliloti nifH promoter to study the spatial proximity relationships. This 88-bp heteroduplex probe (early melted probe) mimics the conformation of the promoter DNA in the closed promoter complex (Fig. 1a) (12), and ⁵⁴ binds to the early melted probe much better than to the homoduplex probe (12). The early melted probe allows assay of ⁵⁴-DNA interactions in the absence of the core RNAP as well as ⁵⁴-DNA interactions within promoter complexes formed in the presence of the core RNAP.

⁵⁴-specific DNA Cleavage—As shown in Fig. 2a, reactions conducted with the mock-conjugated Cys(–) ⁵⁴ variant resulted in no discernible cutting of template and nontemplate strand DNA (compare lanes 1 and 2 and data not shown). The ⁵⁴ variant harboring FeBABE at position 46 in Region I cleaved the template strand DNA between positions –8 and –2 (Fig. 2a, lane 4) and the nontemplate strand DNA between –8 and –5 (data not shown). This result extends previous studies demonstrating that Region I of ⁵⁴ is involved in –12 promoter region proximal interactions (12, 32, 35–37). The ⁵⁴ variant harboring FeBABE at the C-terminal end of the RpoN box (Cys^{463 54}) cut the template strand DNA at two positions: strong cleavage between –30 and –25 (site I) and weaker cleavage between –20 and –16 (site II) (Fig. 2a, lane 10). We tried to minimize the possibilities of any nonspecific DNA cleavage by FeBABE-conjugated Cys^{463 54} by (i) adding heparin to the reactions prior to initiating cleavage, (ii) reducing the cleavage time, and (iii) gel-isolating the cleaved binary FeBABE-conjugated Cys^{463 54}-early melted probe complex prior to analysis by denaturing PAGE. In all cases, the results repeatedly showed that FeBABE-conjugated Cys^{463 54}-mediated cleavage occurred at two positions (sites I and II) on the template strand (Fig. 2a, lane 10 and data not shown). The centers of cleavage sites I and II are separated by a distance of 34 Å on linear B-DNA (assuming a 3.4 Å separation distance between adjacent bases) and are therefore well beyond the FeBABE cleavage range (maximum 12–16 Å). The double cleavage pattern suggests that positions –30 to –25 (site I) and –20 to –16 (site II) are proximal to the RpoN box residue Glu⁴⁶³ within the ⁵⁴-early melted probe complex due to a nonnative conformation of the early melted probe (see below). On the nontemplate strand DNA, FeBABE-conjugated Cys⁴⁶³-mediated cleavage occurred between positions –29 and –26 (data not shown). Overall, this result provides the first physical evidence for the proximity of an RpoN box residue in ⁵⁴ to the consensus –24 promoter region.

View larger version (71K): [in this window] [in a new window]   FIG. 2. FeBABE cleavage of S. meliloti nifH early melted probe by FeBABE-conjugated 54 variants. a, FeBABE cleavage profiles of S. meliloti nifH template strand by 54 Cys(–) (lanes 1 and 2), Cys46 (lanes 3 and 4), Cys455 (lanes 5 and 6), Cys460 (lanes 7 and 8), Cys463 (lanes 9 and 10), and Cys474 (lanes 11 and 12). Reactions to which ascorbate and hydrogen peroxide were added to initiate cleavage are marked with a plus sign; control reactions to which no ascorbate or hydrogen peroxide were added are marked with a minus sign. Lane M contains a mixture of end-labeled S. meliloti nifH promoter DNA fragments as a molecular mass marker. b, activities of the FeBABE-conjugated 54 variants to bind the S. meliloti nifH early melted probe as judged by native PAGE. The migration positions of the free early melted probe and the 54-early melted probe complexes are as indicated.

No discernible DNA cleavage of either DNA strand was seen in complexes containing FeBABE conjugated to Cys⁴⁵⁵, Cys⁴⁶⁰, and Cys^{474 54} variants (Fig. 2a, lanes 6, 8, and 12, and data not shown). We analyzed by native PAGE whether the FeBABE-conjugated Cys⁴⁵⁵, Cys⁴⁶⁰, and Cys^{474 54} variants bind the early melted probe. As shown in Fig. 2b, ⁵⁴ with FeBABE conjugated to Cys⁴⁵⁵ (located N-terminal to the RpoN box) and Cys⁴⁶⁰ (located in the middle of the RpoN box) failed to bind the early melted probe efficiently (lanes 3 and 4, respectively). Likewise, no early melted probe binding was seen with unconjugated Cys⁴⁵⁵ and Cys^{460 54} variants (data not shown), suggesting that the presence of a FeBABE molecule per se is not the cause for the DNA binding defect. In contrast, the FeBABE-conjugated Cys^{474 54} bound the early melted probe well (Fig. 2b, lane 6), demonstrating that residue Lys⁴⁷⁴ is not needed for binding to the ⁵⁴-early melted probe complex and indicating that residue Lys⁴⁷⁴ is not proximal to the promoter.

⁵⁴-RNAP-specific DNA Cleavage—We examined the influence of the core RNAP on promoter cleavage by FeBABE-conjugated ⁵⁴ variants. Initially, we confirmed by native PAGE that all FeBABE-conjugated ⁵⁴ variants formed ⁵⁴-RNAP and promoter complexes on the early melted probe efficiently (data not shown). No discernible cleavage was seen with FeBABE-conjugated Cys(–) ⁵⁴-RNAP on either DNA strands (Fig. 3a, lane 2 and data not shown). The DNA cleavage pattern by FeBABE-conjugated Cys⁴⁶ and Cys^{463 54} variants was largely unchanged in the presence of core RNAP (compare Fig. 2a, lanes 4 and 10, with Fig. 3a, lanes 4 and 11, respectively). Strikingly, DNA cleavage was seen with FeBABE-conjugated Cys⁴⁵⁵, Cys⁴⁶⁰, and Cys^{474 54} variants in the presence of the core RNAP. FeBABE-modified ⁵⁴ Cys⁴⁵⁵-RNAP cut the template strand DNA weakly between positions –24 and –21 (Fig. 3a, lane 6, marked with a dotted line), but no DNA cleavage was detected on the nontemplate strand (data not shown). Similarly, FeBABE-conjugated ⁵⁴ Cys⁴⁷⁴-RNAP cut the template strand DNA between positions –32 and –28 (Fig. 3a, lane 13) but not the nontemplate strand DNA (data not shown). The detection of DNA cleavage by FeBABE-conjugated Cys^{474 54} only in the presence of core RNAP suggests that core RNAP-dependent conformational changes in ⁵⁴ lead to changes in the spatial relationship between residue Lys⁴⁷⁴ and DNA. The DNA binding defects of the FeBABE-conjugated Cys⁴⁵⁵ and Cys^{460 54} variants indicate that core RNAP merely functions to increase their binding to the early melted probe.

View larger version (36K): [in this window] [in a new window]   FIG. 3. FeBABE cleavage of S. meliloti nifH early melted probe by 54-RNAP reconstituted with FeBABE-conjugated 54 variants. a, FeBABE cleavage profiles of S. meliloti nifH template strand by 54 Cys(–) (lanes 1 and 2), Cys46 (lanes 3 and 4), Cys455 (lanes 5 and 6), Cys460 (lanes 7–9), Cys463 (lanes 10 and 11), and Cys474 (lanes 12 and 13) RNAPs. Reactions to which ascorbate and hydrogen peroxide were added to initiate cleavage are marked with a plus sign, control reactions to which no ascorbate or hydrogen peroxide were added are marked with a minus sign. Lane M contains a mixture of end-labeled S. meliloti nifH promoter DNA fragments as a molecular mass marker. In lane 9, 54 Cys460-RNAP-early melted probe complexes were isolated from a native gel (see "Experimental Procedures") prior to analysis by denaturing PAGE. In lane 6, the dotted line indicates weak DNA cleavage by FeBABE-conjugated 54 Cys455-RNAP. b, PhosphorImager traces (shown here for the template strand) of 54 Cys455-RNAP-, Cys460-RNAP-, and Cys463-RNAP-promoter complexes on the early melted probe following treatment with DNase I (black lines). The Phosphor-Imager traces of free DNA (light gray lines) and wild-type 54-RNAP-early melted probe complexes (gray lines) following treatment with DNase I are shown for comparison. The DNA sequence (from position –34 to –5) that becomes footprinted by 54-RNAP is shown, and the location of the –24 GG-region is indicated. The asterisks indicate sites where differences are seen in the footprints of the 54 Cys460-RNAP and wild-type 54-RNAP on the early melted probe.

Interestingly, FeBABE-conjugated ⁵⁴ Cys⁴⁶⁰-RNAP cut the template strand DNA at three distinct sites: strong cleavage between –19 and –16 (site III) and weaker cleavage between –28 and –26 (site IV) and between –8 and –5 (site V). On the nontemplate strand DNA, FeBABE-conjugated ⁵⁴ Cys⁴⁶⁰-RNAP cut strongly between positions –19 and –16 and weakly between –28 and –25 (data not shown). On the template strand DNA, ⁵⁴ Cys⁴⁶⁰-RNAP cleavage sites III and IV overlap ⁵⁴ Cys⁴⁶³-RNAP cleavage site II (Fig. 3a, lane 11; between positions –17 and –15) and site I (Fig. 3a, lane 11; between positions –28 and –24), respectively. Since the centers of ⁵⁴ Cys⁴⁶⁰-RNAP and Cys⁴⁶³-RNAP cleavage at sites I and II (for FeBABE-conjugated ⁵⁴ Cys⁴⁶³-RNAP-mediated cleavage) and III and IV (for FeBABE-conjugated ⁵⁴ Cys⁴⁶⁰-RNAP-mediated cleavage) are separated by 10 base pairs, 34 Å on linear B-DNA, and so are beyond the FeBABE cleavage range, we suggest that the DNA is kinked at the –24 consensus promoter region, causing sites I/IV and II/III to be in close proximity to each other. The intensities of the cleavage by ⁵⁴ Cys⁴⁶⁰-RNAP and ⁵⁴ Cys⁴⁶³-RNAP suggest that within ⁵⁴-RNAP-early melted probe complexes, residue Lys⁴⁶⁰ is orientated away from the –24 GG-region and is facing DNA downstream of the –24 GG-region, whereas Glu⁴⁶³ is orientated toward the –24 GG-region. The DNA cleavage at site V by FeBABE-conjugated ⁵⁴ Cys460-RNAP was consistently observed (Fig. 3a, lane 8) and was seen under conditions where the probability of nonspecific cleavage was minimized (see above; Fig. 3a, lane 9). DNA cleavage at site V could have occurred due to an artifactual trajectory of the DNA within ⁵⁴ Cys⁴⁶⁰-RNAP-early melted probe complexes or because of nonnative ⁵⁴ conformation. Analysis of the ⁵⁴ Cys⁴⁶⁰-RNAP early melted probe complexes by DNase I footprinting indicated that the interaction Cys⁴⁶⁰-RNAP makes with the early melted probe is different when compared with FeBABE-conjugated ⁵⁴ Cys⁴⁵⁵-RNAP, Cys⁴⁶³-RNAP, or the wild-type ⁵⁴-RNAP (Fig. 3b), consistent with the early melted probe binding defect seen with the Cys^{460 54} in the absence of core RNAP (Fig. 2b, lane 4).

RpoN Box Residue 463 Is Proximal to the Consensus –24 GG-region of ⁵⁴-dependent Promoter Complexes
We explored the proximity of residue Glu⁴⁶³ in the RpoN box of ⁵⁴ to the consensus –24 GG-region of ⁵⁴-dependent promoter complexes by conducting the proximity cleavage experiments on linear and supercoiled S. meliloti nifH promoter homoduplex DNA. Proximity-based cleavage of linear 88-bp S. meliloti nifH homoduplex promoter probe by FeBABE-conjugated ⁵⁴ Cys⁴⁶³-RNAP revealed strong cleavage in the template strand between positions –29 and –26 and weak cleavage at positions –18 and –17 (Fig. 4a, lane 4). The nontemplate strand DNA was strongly cut between positions –29 and –26 (data not shown). Similarly, FeBABE-conjugated ⁵⁴ Cys⁴⁶³-RNAP cut the template strand DNA at positions –27, –26, and –24 (strong cleavage) and at positions –16 and –17 (weak cleavage) within closed promoter complexes formed on the supercoiled S. meliloti nifH promoter on pMKC28 (Fig. 4b, lane 4). The nontemplate strand DNA was cut at positions –30, –29, –22, and –21 (see below). Thus, cleavage profiles on three different S. meliloti nifH promoter variants (early melted probe, homoduplex probe, and pMKC28) establish the physical proximity of the RpoN box residue Glu⁴⁶³ to the consensus –24 promoter region.

View larger version (29K): [in this window] [in a new window]   FIG. 4. FeBABE cleavage of S. meliloti nifH homoduplex probe by 54-RNAP reconstituted with FeBABE-conjugated 54 Cys463. a, FeBABE cleavage profile of S. meliloti nifH template strand by 54 Cys(–)-RNAP (lanes 1 and 2) and 54 Cys463-RNAP (lanes 3 and 4) on the linear 88-base pair homoduplex probe. b, FeBABE cleavage profile of S. meliloti nifH template strand by 54 Cys(–)-RNAP (lanes 1 and 2) and 54 Cys463-RNAP (lanes 3 and 4) on the supercoiled plasmid pMKC28. Reactions to which ascorbate and hydrogen peroxide were added to initiate cleavage are marked with a plus sign, and control reactions to which no ascorbate or hydrogen peroxide were added are marked with a minus sign. In a, lane M contains a mixture of end-labeled S. meliloti nifH promoter DNA fragments as a molecular mass marker. In b, lanes marked A, C, G, and T contain chain termination DNA sequencing reactions conducted with pMKC28 and the nucleotide analogues ddATP, ddCTP, ddGTP, and ddTTP, respectively. The relevant DNA sequence is shown with the consensus GG- and GC-regions marked in boldface type. The black dots indicate the positions where FeBABE-mediated cleavage occurs.

Contribution of the Core RNAP to ⁵⁴ Interactions at the Consensus –12 and –24 Promoter Elements
We used the FeBABE-conjugated Cys⁴⁶ and Cys^{463 54} variants to further study ⁵⁴ interactions with the consensus –24 and –12 promoter regions because both (⁵⁴ variants were active for transcription in vitro and gave strong and distinct cleavage patterns on both DNA strands of the early melted probe. Cleavage reactions were conducted on the S. meliloti nifH promoter on pMKC28 or the S. meliloti nifH homoduplex promoter probe in the absence and presence of the core RNAP.

DNA Cleavage by FeBABE-conjugated Cys^{46 54}—As shown in Fig. 5a, no cleavage is seen on either DNA strand with FeBABE-conjugated Cys^{46 54} in the absence of core RNAP on the S. meliloti nifH promoter on pMKC28 (lanes 1 and 2; data not shown). Strikingly, in the presence of core RNAP, FeBABE-conjugated Cys^{46 54} cleaved the nontemplate strand DNA between positions –10 and –8 (Fig. 5a, lane 3). No template strand DNA cleavage was detected by the FeBABE-conjugated Cys^{46 54} even in the presence of core RNAP (see below). The addition of heparin to the ⁵⁴ Cys⁴⁶-RNAP promoter complex prior to initiating cleavage, in order to dissociate the core RNAP from ⁵⁴ (38) resulted in the disappearance of the nontemplate strand cleavage between positions –10 and –8 (Fig. 5a, lane 4). DNase I footprinting that followed heparin treatment showed that Cys^{46 54} remained bound to the promoter (Fig. 5b). Similarly, nontemplate strand DNA cleavage by FeBABE-conjugated Cys^{46 54} was dependent on core RNAP within promoter complexes formed on the linear S. meliloti nifH homoduplex probe (Fig. 5c, compare lanes 2 and 3). No template strand cleavage by FeBABE-conjugated Cys^{46 54} was detectable within the promoter complex formed on the homoduplex probe (data not shown).

View larger version (33K): [in this window] [in a new window]   FIG. 5. FeBABE cleavage of S. meliloti nifH promoter probes by FeBABE-conjugated Cys46 and Cys463 in the presence and absence of core RNAP. a, FeBABE cleavage profiles (shown here for the nontemplate strand) on pMKC28 by FeBABE-conjugated 54 Cys46 (lanes 1 and 2) and 54 Cys46-RNAP (lanes 3 and 4). b, PhosphorImager traces (shown here for the nontemplate strand) of FeBABE-conjugated 54 Cys46 and wild-type 54 footprints on pMKC28 before (gray lines) and after heparin challenge (+H; black lines) followed by DNase I treatment under the condition shown in a, lanes 1 and 2, respectively, but in the absence of ascorbate and hydrogen peroxide. The PhosphorImager traces of free DNA (pMKC28) following DNase I treatment are shown for comparison (light gray lines). The DNA sequence (from position –34 to –5) that becomes footprinted by 54 is shown, and the location of the –12 and –24 GC- and GG-regions, respectively, are indicated. c, FeBABE cleavage profile of S. meliloti nifH nontemplate strand by 54 Cys46 (lanes 1 and 2) and 54 Cys46-RNAP (lanes 3 and 4) on the linear 88-bp homoduplex probe. In lane 2, the dotted line indicates the DNA cleavage site. d, FeBABE cleavage profiles (shown here for the nontemplate strand) on pMKC28 by FeBABE-conjugated 54 Cys463 and Cys463-RNAP. In a and d, lanes marked A, C, G, and T contain chain termination DNA sequencing reactions conducted with pMKC28 and the nucleotide analogues ddATP, ddCTP, ddGTP, and ddTTP, respectively. The relevant DNA sequence is shown with the consensus GG- and GC-regions marked in boldface type. The black dots indicate the positions where FeBABE-mediated cleavage occurs. In c, lane M contains a mixture of end-labeled S. meliloti nifH promoter DNA fragments as a molecular mass marker. The minus and plus signs indicate the absence and presence of ascorbate and hydrogen peroxide in the reactions.

View larger version (24K): [in this window] [in a new window]   FIG. 5. FeBABE cleavage of S. meliloti nifH promoter probes by FeBABE-conjugated Cys46 and Cys463 in the presence and absence of core RNAP. a, FeBABE cleavage profiles (shown here for the nontemplate strand) on pMKC28 by FeBABE-conjugated 54 Cys46 (lanes 1 and 2) and 54 Cys46-RNAP (lanes 3 and 4). b, PhosphorImager traces (shown here for the nontemplate strand) of FeBABE-conjugated 54 Cys46 and wild-type 54 footprints on pMKC28 before (gray lines) and after heparin challenge (+H; black lines) followed by DNase I treatment under the condition shown in a, lanes 1 and 2, respectively, but in the absence of ascorbate and hydrogen peroxide. The PhosphorImager traces of free DNA (pMKC28) following DNase I treatment are shown for comparison (light gray lines). The DNA sequence (from position –34 to –5) that becomes footprinted by 54 is shown, and the location of the –12 and –24 GC- and GG-regions, respectively, are indicated. c, FeBABE cleavage profile of S. meliloti nifH nontemplate strand by 54 Cys46 (lanes 1 and 2) and 54 Cys46-RNAP (lanes 3 and 4) on the linear 88-bp homoduplex probe. In lane 2, the dotted line indicates the DNA cleavage site. d, FeBABE cleavage profiles (shown here for the nontemplate strand) on pMKC28 by FeBABE-conjugated 54 Cys463 and Cys463-RNAP. In a and d, lanes marked A, C, G, and T contain chain termination DNA sequencing reactions conducted with pMKC28 and the nucleotide analogues ddATP, ddCTP, ddGTP, and ddTTP, respectively. The relevant DNA sequence is shown with the consensus GG- and GC-regions marked in boldface type. The black dots indicate the positions where FeBABE-mediated cleavage occurs. In c, lane M contains a mixture of end-labeled S. meliloti nifH promoter DNA fragments as a molecular mass marker. The minus and plus signs indicate the absence and presence of ascorbate and hydrogen peroxide in the reactions.

The core RNAP-dependent DNA cleavage of the supercoiled S. meliloti nifH promoter (Fig. 5a) and the linear homoduplex probe (Fig. 5c) by FeBABE-conjugated Cys^{46 54} is notable because FeBABE-conjugated Cys^{46 54} cleaved both DNA strands of the early melted probe, in the absence and in the presence of core RNAP (Fig. 2a, lane 4, and Fig. 3a, lane 4; data not shown). Thus, it seems that the DNA fork junction structure at the –12 position can substitute for core RNAP. We suggest that core RNAP-directed conformational changes in ⁵⁴ place residue Leu⁴⁶ in Region I proximal to the consensus –12 GC-region within closed promoter complexes, a result fully consistent with the requirement for ⁵⁴ and the core RNAP subunits to generate the DNA distortion next to the consensus –12 GC-region (26).

DNA Cleavage by FeBABE-conjugated Cys^{463 54}—In contrast to Region I residue Leu⁴⁶, promoter DNA conformation or the presence or absence of core RNAP appeared to have no influence on the proximity between the RpoN box residue Glu⁴⁶³ and the consensus –24 GG-region. As shown in Fig. 5d, FeBABE-conjugated Cys^{463 54} and the ⁵⁴ Cys⁴⁶³-RNAP cut the nontemplate strand DNA of the S. meliloti nifH promoter on pMKC28 at positions –30, –29, –22, and –21 both in the presence and absence of core RNAP (lanes 1–4), suggesting that some RpoN box interactions with the consensus –24 GG-region that are dominant for overall DNA binding by ⁵⁴-RNAP are independent of core RNAP subunits and the –12 GC-region DNA structure.

The Spatial Relationship between ⁵⁴-RNAP and the Consensus –24 Region Remains Unchanged during Transcription Initiation at ⁵⁴-dependent Promoters
The ⁵⁴-RNAP interactions near the –12 promoter region, where DNA melting originates, are believed to be subject to conformational changes (12, 35, 36, 39, 40–42). To investigate whether the proximity relationship between the RpoN box residue Glu⁴⁶³ and the –24 promoter region changes during open complex formation and to seek physical evidence that the proximity relationship between Region I residue Leu⁴⁶ and the –12 promoter region are subject to change during open complex formation, we conducted DNA cleavage experiments on closed and open promoter complexes formed by the FeBABE-conjugated ⁵⁴ Cys⁴⁶³-RNAP and ⁵⁴ Cys⁴⁶-RNAP using pMKC28 as template DNA. We also studied promoter complexes that had initiated transcription.

⁵⁴ Cys⁴⁶³-RNAP—As shown in Fig. 6a, no detectable changes in the cleavage pattern on either DNA strand were seen within closed (lanes 2 and 10), open (lanes 4 and 12), and initiated (lanes 6 and 14) promoter complexes formed by the ⁵⁴ Cys⁴⁶³-RNAP. We considered the possibility that the ⁵⁴ Cys⁴⁶³-RNAP closed complex might not have fully isomerized to form the open or the initiated promoter complex under the conditions used. In contrast to closed promoter complexes, transcription-competent open and initiated ⁵⁴-RNAP promoter complexes are heparin-stable. The addition of heparin did not result in any detectable changes in the cleavage patterns on both DNA strands (Fig. 6a, lanes 7, 8, 15, and 16). We also used DNase I footprinting to prove that the ⁵⁴ Cys⁴⁶³-RNAP closed promoter complexes have indeed isomerized to form the open and initiated complexes in response to activation under the conditions used. As shown in Fig. 6b, the ⁵⁴ Cys⁴⁶³-RNAP footprint on the DNA is extended toward the transcription start site in the open and initiated complex reactions, indicating isomerization of the ⁵⁴ Cys⁴⁶³-RNAP closed complex in response to activation. The bona fide nature of the heparin-resistant open and initiated promoter complexes formed by the ⁵⁴ Cys⁴⁶³-RNAP under the cleavage conditions was further confirmed by their ability to produce transcripts upon the addition of the remaining nucleotides following the heparin challenge (data not shown). We conclude that residue Glu⁴⁶³ in ⁵⁴ does not change its location with respect to DNA during open complex formation and transcription initiation.

⁵⁴ Cys⁴⁶-RNAP—As shown in Fig. 6c, FeBABE-conjugated ⁵⁴ Cys⁴⁶-RNAP cleaves the nontemplate strand DNA strongly between positions –10 and –8 (lane 2), but no cleavage is detected on the template strand DNA. In assays lacking heparin, conditions for open or initiated complex formation did not change the cleavage patterns (Fig. 6c, lanes 4, 6, 12, and 14). The addition of heparin after open and/or initiated complex formation, a condition that is expected to destroy residual or partly isomerized closed complexes, led to the disappearance of DNA cleavage on both DNA strands (Fig. 6c, lanes 7, 8, 15, and 16). We confirmed by DNase I footprinting and in vitro transcription that the ⁵⁴ Cys⁴⁶-RNAP had formed productive open and initiated complexes following the heparin challenge (Fig. 6d and data not shown). Additional DNA cleavage experiments using a ⁵⁴ variant harboring FeBABE at positions Cys⁴⁶ in Region I and Cys⁴⁶³ in the RpoN box² confirmed that the spatial relationship between the RpoN box residue Glu⁴⁶³ and the consensus –24 promoter region remain unchanged during the transition of the closed promoter complex to a transcription-competent open complex (Fig. 6e, compare lanes 2 and 5). In contrast, the –12 promoter region interactions changed during open complex formation (Fig. 6e, compare lanes 2 and 5), consistent with the –12-⁵⁴-RNAP interactions being a target for the remodeling activity of ⁵⁴-dependent activators (11, 12, 27, 39, 40).

View larger version (30K): [in this window] [in a new window]   FIG. 6. 54-RNAP-DNA interactions with the consensus –24 promoter region of 54-dependent promoter remain unchanged during open complex formation and transcription initiation. a, template and nontemplate strand cleavage by FeBABE-conjugated 54 Cys463-RNAP within the closed (lanes 1, 2, 9, and 10), open (lanes 3, 4, 7, 11, 12, and 15), and initiated (lanes 5, 6, 8, 13, 14, and 16) promoter complexes formed on pMKC28. b, PhosphorImager traces (shown here for the template strand) of closed, open, and initiated promoter complexes (black lines) formed by the Cys463-RNAP on pMKC28 under the cleavage conditions shown in a, lanes 10, 15, and 16, respectively, but in the absence of ascorbate and hydrogen peroxide. The DNA sequence (from position –34 to –5) that becomes footprinted by 54-RNAP is shown, and the location of the –12 and –24 GC- and GG-regions, respectively, are indicated. The DNA sequence that becomes protected only in open and initiated promoter complexes is shown by the dotted line. The free DNA that has been subjected to DNase I cleavage is shown in gray. c, template and nontemplate strand cleavage by 54 Cys46-RNAP within the closed (lanes 1, 2, 9, and 10), open (lanes 3, 4, 7, 11, 12, and 15), and initiated (lanes 5, 6, 8, 13, 14, and 16) promoter complexes formed on pMKC28. d, PhosphorImager traces (shown here for the template strand and annotated as in b) of closed, open, and initiated promoter complexes formed by 54 Cys46-RNAP on pMKC28 under the cleavage conditions shown in c, lanes 10, 15, and 16, respectively, but in the absence of ascorbate and hydrogen peroxide. In a and c, lanes marked A, C, G, and T contain chain termination DNA sequencing reactions conducted with pMKC28 and the nucleotide analogues ddATP, ddCTP, ddGTP, and ddTTP, respectively. The relevant DNA sequence is shown with the consensus GG- and GC-regions marked in boldface type. The black dots indicate the positions where FeBABE-mediated cleavage occurs. In a, the white dot indicates weak cleavage seen at positions –26 only in the presence of heparin. e, nontemplate strand DNA cleavage by 54-RNAP reconstituted with 54 variant harboring FeBABE at positions Leu46 and Glu463 demonstrating that the relationship between 54 Region I residue Leu46 and the consensus –12 promoter region has changed in open (lane 5) promoter complexes formed on pMKC28, whereas the relationship between the 54 RpoN box residue Glu463 and the consensus –24 promoter region remains unchanged. The dotted line in lane 5 indicates the absence of DNA cleavage at the –12 position. The figure is annotated as in a and c.

The Spatial Relationship between ⁵⁴ and the –24 Promoter Region Changes during Early Elongation
The observation that the spatial relationship between some parts of the RpoN box in ⁵⁴-RNAP and the consensus –24 promoter region remains unchanged upon open complex formation and transcription initiation prompted us to investigate whether ⁵⁴ interactions with the –24 promoter region would change during early elongation, where more than 3 nucleotides of RNA has been synthesized. As schematically outlined in Fig. 7, we made early elongating promoter complexes on pMKC28 with the FeBABE-conjugated ⁵⁴ Cys⁴⁶³-RNAP under conditions that resulted in the synthesis of an RNA product that was more than 3 nucleotides (18) and probably 9 nucleotides in length (Cys⁴⁶³-RNAP_EC).

Reaction I—As shown in Fig. 7a, the characteristic FeBABE-conjugated ⁵⁴ Cys⁴⁶³-RNAP-mediated DNA cutting near the consensus –24 promoter region was not detected either on the nontemplate strand (lane 2) or the template strand (data not shown) DNA within Cys⁴⁶³-RNAP_EC. Since DNA cleavage at the –24 promoter region was still detected on both DNA strands within initiated promoter complexes formed with the FeBABE-conjugated ⁵⁴ Cys⁴⁶³-RNAP where an RNA trimer had formed (Fig. 6a, lanes 8 and 16), we suggest that the synthesis of more than 3 and probably 9 nucleotides of RNA leads to changes in the relationship between the RpoN box residue Glu⁴⁶³ and the –24 promoter region. Thus, the absence of DNA cleavage at the –24-position within Cys⁴⁶³-RNAP_EC indicates that the spatial relationship between the RpoN box residue Glu⁴⁶³ and the consensus –24 region has changed during early elongation.

Reaction II—To determine whether the ⁵⁴ Cys⁴⁶³-RNAP_EC complex was productive for transcription, we added [-³²P]UTP, which was missing from the reaction during the formation of Cys⁴⁶³-RNAP_EC, to reaction II. Analysis of reaction II by denaturing PAGE confirmed that the stalled complex was productive for specific transcription from pMKC28 (Fig. 7b). Further, equal amounts of transcripts were produced from initiated promoter complexes formed with the FeBABE-conjugated ⁵⁴ Cys⁴⁶³-RNAP (where DNA cleavage at the –24 promoter region was detected, upon the addition of the remaining nucleotides following the heparin challenge (Fig. 6a) and ⁵⁴ Cys⁴⁶³-RNAP_EC (Fig. 7, a and c). This result suggests that the lack of DNA cleavage at the –24-position within Cys⁴⁶³-RNAP_EC is not due to the number of Cys⁴⁶³-RNAP_EC being significantly less than the number of initiated FeBABE-conjugated ⁵⁴ Cys⁴⁶³-RNAP-promoter complexes under the conditions used here.

Additional control assays in which core RNAP was omitted from the reactions with FeBABE-conjug