Organization of Open Complexes at Escherichia coli Promoters
LOCATION OF PROMOTER DNA SITES CLOSE TO REGION 2.5 OF THE sigma 70 SUBUNIT OF RNA POLYMERASE*

Jonathan A. BownDagger , Jeffrey T. Owens§, Claude F. Meares§, Nobuyuki Fujita, Akira Ishihama, Stephen J. W. BusbyDagger , and Stephen D. MinchinDagger parallel

From the Dagger  School of Biochemistry, The University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom, § Department of Chemistry, University of California, Davis, California 95616, and  Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411, Japan

    ABSTRACT
Top
Abstract
Introduction
References

A cysteine-tethered DNA cleavage agent has been used to locate the position of region 2.5 of sigma 70 in transcriptionally competent complexes between Escherichia coli RNA polymerase and promoters. In this study we have engineered sigma 70 to introduce a unique cysteine residue at a number of positions in region 2.5. Mutant proteins were purified, and in each case, the single cysteine residue used as the target for covalent coupling of the DNA cleavage agent p-bromoacetamidobenzyl-EDTA·Fe (FeBABE). RNA polymerase core reconstituted with tagged sigma  derivatives was shown to be transcriptionally active. Hydroxyl radical-based DNA cleavage mediated by tethered FeBABE was observed for each derivative of RNA polymerase in the open complex. Our results show that region 2.5 is in close proximity to promoter DNA just upstream of the -10 hexamer. This positioning is independent of promoter sequence. A model for the interaction of this region of sigma  with promoter DNA is discussed.

    INTRODUCTION
Top
Abstract
Introduction
References

Promoter recognition requires sequence-specific contacts by the transcriptional apparatus. At most promoters these contacts are made upstream from the transcription start point. Once the transcriptional apparatus has bound the promoter to form a closed complex, an isomerization event occurs to generate the open complex, forming the single-stranded template required for transcription. The bacterium Escherichia coli provides a good model for understanding protein-DNA interactions during transcription initiation. E. coli uses a single core RNA polymerase for transcription elongation with subunit composition alpha 2beta beta '. Promoter specificity is principally afforded by a separate subunit, sigma , which associates with the core enzyme to give holoenzyme (RNAP)1 but dissociates once sequence-specific promoter DNA contacts are no longer required (1). The sigma 70 subunit, encoded by rpoD, is one of several sigma  subunits utilized by E. coli and is responsible for directing the transcription of most genes during vegetative growth. RNAP is capable of sequence-specific transcription initiation in the absence of other transcription factors. Factor-independent transcription is reliant on the ability of sigma 70 to make stable contacts with the promoter DNA (1, 2). E. coli promoters contain two very conserved motifs, the -10 and -35 hexamers (3), and several less-conserved sequences including the UP element (4) and the extended -10 motif (5). The extended -10 motif (5'-TGXTATAAT-3') can drive factor-independent transcription at several bacterial promoters lacking homology to the consensus within the -35 region (6, 7). Therefore this TGX motif is able to compensate for a poor -35 hexamer. The TG motif has been shown to be important for promoter activity in several other bacterial species (8-11). Work from many laboratories has defined the regions within RNA polymerase that are responsible for sequence-specific contacts within promoter DNA. Regions 2.4 and 4.2 of sigma 70 contact the -10 and -35 hexamers, respectively (1, 2), whereas the C-terminal domain of the alpha  subunit (alpha CTD) contacts the UP element (4). Recent work from this laboratory has indicated that a newly defined region of sigma 70, region 2.5, is responsible for making sequence-specific contacts with the extended -10 motif (12). This region was identified by screening for altered or relaxed specificity mutants of sigma 70 capable of compensating for down-mutations within the extended -10 motif. One relaxed specificity mutant, sigma 70 E458G, was isolated (12). The E458G substitution partially suppressed the effect of changing the G·C base pair of the TG motif, suggesting a role for the side chain at position 458 in contacting the extended -10 motif.

The aim of the study presented in this paper is to complement the genetic study with biophysical data to support the suggested role of region 2.5. We wanted to show that, in open, transcriptionally competent complexes at E. coli promoters, region 2.5 of sigma 70 is located near to promoter DNA, just upstream of the -10 hexamer. To do this, we exploited a novel method that relies on tethering a DNA cleavage agent to a single specific amino acid side chain of a protein (13). The reagent p-bromoacetamidobenzyl-EDTA·Fe (FeBABE) has one reactive group facilitating covalent attachment to cysteine side chains, whereas a second group holds a single metal atom in a tight coordination complex (14). Under appropriate conditions, the divalent cation can participate in the generation of hydroxyl radicals, which attack deoxyribose units, resulting in DNA strand scission (15). Recently, this chemistry has been applied to the study of the interaction of alpha CTD of E. coli RNAP with promoter DNA. Hydroxyl radical DNA cleavage mediated by FeBABE showed that the two alpha CTD subunits are arranged asymmetrically, contacting different halves of the UP element and that activator contact patches are available on both subunits (16, 17). To identify sites on promoter DNA that are near to region 2.5 of sigma 70 in open complexes, amino acids in this region were replaced with cysteine for conjugation with the DNA cleavage agent FeBABE.

    EXPERIMENTAL PROCEDURES

Strains and Materials-- E.coli strain DH5alpha (supE44Delta lacU169(phi 80 lacZDelta M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1) was used for all cloning (18). Unless indicated otherwise, chemicals were purchased from Sigma, radiochemicals were from NEN Life Science Products, synthetic oligonucleotides were from Alta Bioscience at the University of Birmingham, restriction and DNA-modifying enzymes were from New England Biolabs and Taq DNA polymerase from Boehringer Mannheim.

Plasmids Encoding sigma 70-- Mutants of rpoD encoding a single cysteine were generated by megaprimer polymerase chain reaction (19) using plasmid pGEMD(S), which contains a cysteine-free rpoD gene as a template (20, 21). Mutagenic oligonucleotides complementary to the template strand of rpoD used were CYS454 (5'-CCATCCGTATTCCGTGTCACATGATTGAGACCATC-3'), CYS459 (5'-GTGCATATGATTGAGTGTATCAACAAGCTCAAC-3'), and CYS461 (5'-TGATTGAGACCATCTGTAAGCTCAACCGTATT-3'), encoding mutations corresponding to cysteine substitutions at positions 454, 459, and 461 of sigma 70. In the first round of polymerase chain reaction, a mutagenic oligonucleotide and oligonucleotide D13346 (5'-GGTCGCAGAATCCAGCGGC-3'), annealing to the coding strand of rpoD were used to amplify a "megaprimer" fragment of rpoD. In the second round of polymerase chain reaction, the megaprimer and oligonucleotide D12444 (5'-GCAGATTAATGATATCAACCGTCGT-3') annealing to the template strand of rpoD were used to amplify a 540-base pair fragment of rpoD. Plasmid cloning vector Pinpoint-Xa1 (Promega) was used for direct cloning of polymerase chain reaction products according to the manufacturer's instructions, and the recombinants were used for sequencing. A cysteine substitution at position 458 had previously been constructed (22). Restriction enzymes XhoI and BamHI were used to subclone internal fragments of rpoD containing the desired mutations into pGEMD(S) (21). Construction of the other single cysteine mutants used (422C and 581C) are described elsewhere (21).

Promoter Fragments-- The KAB-TTcon (23) and KAB-TG (24) promoters were cloned on EcoRI-HindIII fragments in the galK fusion vector pAA121. The promoter galP1(19T8A9A) (25) was cloned as an EcoRI-HindIII fragment in pBR322. The sequence of the three promoters is shown in Fig. 1. For FeBABE cleavage reactions and transcription run-off assays, 850-base pair PstI-HindIII fragments were prepared from plasmid DNA purified on caesium chloride gradients. In addition to the promoter under study, these fragments also contained two additional promoters, pX and pbla. For the FeBABE cleavage analysis, the template strand was labeled at the HindIII end with [gamma -32P]ATP and T4 polynucleotide kinase or the nontemplate strand was labeled at the HindIII end using [alpha -32P]dATP and the E. coli DNA polymerase Klenow fragment.


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Fig. 1.   The nucleotide sequence of three promoters used in this work; KAB-TG, KAB-TTcon, and galP1(19T8A9A). The promoter consensus recognized by the E. coli sigma 70 holoenzyme is also shown. The -35 and extended -10 sequence elements are underlined.

Proteins-- Single cysteine mutants of sigma 70 were purified by the method described previously for the wild-type protein (20, 26), with the important modification of the exclusion of thiol-containing reducing agents (dithiothreitol or beta -mercaptoethanol) from all steps in purification. Purified mutant subunits were dialyzed against storage buffer (10 mM Tris-HCl, pH 7.6, at 4 °C, 10 mM MgCl2, 0.1 mM EDTA, 100 mM KCl, and 50% glycerol) and stored at -20 °C.

Conjugation of sigma 70 with FeBABE-- FeBABE was synthesized and characterized as described previously (27). Single cysteine derivatives of sigma 70 were used for conjugation with FeBABE based on the method of Murakami et al. (16). Conjugation was initiated by mixing 1.2 ml of 6.67 µM protein solution in conjugation buffer (10 mM Hepes, 200 mM KCl, and 2 mM EDTA, pH of 8.0) with 9 µl of 18 mM FeBABE in Me2SO. After incubation at 37 °C for 4 h, excess unreacted FeBABE was removed by dialysis against storage buffer. The efficiency of conjugation was determined by estimating free side chains of both conjugated and un-conjugated proteins with the fluorescent reagent CPM (7-diethylamino-3-(4'maleimidylphenyl)-4-methylcoumarin) (Molecular Probes) (27).

Reconstitution of RNA Polymerase Holoenzyme-- A 10-fold molar excess of sigma 70 was mixed with core RNAP and incubated at 20 °C for 30 min (26).

DNA Cleavage by FeBABE-- RNAP holoenzyme (300 nM) was mixed with 32P-end-labeled promoter fragment (0.4 nM) in a reaction volume of 35 µl (20 mM HEPES, pH 8.0, 5 mM MgCl2, 50 mM potassium glutamate, 50 µg/ml bovine serum albumin, 5 µg/ml poly(dI·dC)) and incubated at 37 °C for 20 min. Complexes were challenged with heparin (200 µM at 37 °C for 5 min). DNA cleavage was initiated by the addition of sodium ascorbate (2 mM) followed by incubation at 37 °C for 20 min. Modified DNA was extracted with phenol/chloroform and precipitated with ethanol before analysis on a 6% polyacrylamide gel containing M urea. Gels were calibrated with Maxam-Gilbert G+A sequence ladders and were processed and scanned using a PhosphorImager (Molecular Dynamics).

In Vitro Transcription-- The activity of RNAP reconstituted with wild-type, un-conjugated, and conjugated sigma 70 derivatives was measured by in vitro transcription assays (28). Fragments used for cleavage analysis were also used as templates for in vitro transcription. The derivatives of the gal promoter were expected to generate run-off products of 51 nucleotides. DNA template (5 nM) and RNAP holoenzyme (100 nM) were preincubated in 12 µl of transcription buffer (10 mM Tris, pH 8.0, 50 mM NaCl, 2.5 mM MgCl2, 50 µM EDTA, 0.5 mM dithiothreitol, 25 µg/ml bovine serum albumin, 2.5% glycerol) for 5 min at 37 °C. Transcription was initiated by the addition of unlabeled nucleotide triphosphates ATP, CTP, GTP (200 µM), and UTP (10 µM), 0.5 µCi of [alpha -32P]UTP (800 Ci/mmol), 100 µg/ml heparin. Reactions were stopped by the addition of an equal volume of run-off stop mix (20 mM EDTA, 80% deionized formamide, 0.1% bromphenol blue, 0.1% xylene cyanol). Transcripts were analyzed on a 6% polyacrylamide gel containing 6 M urea and scanned using a PhosphorImager.

Modeling of RNA Polymerase-Promoter Interactions-- Modeling of region 2.5 interactions with promoter DNA is based on secondary structure prediction and genetic evidence. Secondary structure prediction suggests that Glu-458 is part of a alpha -helix starting at Val-454. An octapeptide from Val-454 to Asn-461 was constructed using the molecular modeling package Quanta by Molecular Simulations, Inc. and energy minimized to place amino acid side chains in sterically favorable positions. This peptide was then manually docked into the major groove of a model of B-form DNA based on the sequence of KAB-TG from position +1 to -29 with the carboxyl group of Glu-458 making base-pair edge hydrogen bond interactions with N6 of adenine at position -15 and N4 of cytosine at position -14 (i.e. the template strand of the TG motif). In this position, no steric clashes were observed. The model was extended to include the helix of region 2.4 (helix 14) derived from the crystal structure of a tryptic fragment of sigma 70 (29). The atomic coordinates for the fragment (1SIG) were obtained from the Protein Data Bank (Brookhaven National Laboratory, Upton, Long Island2). Genetic studies show that Gln-437 and Thr-440 are involved in interactions with position -12 (30, 31). The coordinates obtained were used to dock helix 14 with residues 437 and 440 in hydrogen-bonding contact with the base pair edge at -12 (see Fig. 7). The minor groove at the center on the -10 element must be placed on the inside of a curvature for efficient promoter recognition by E. coli RNAP (32). In addition, many studies show that recognition of -10 and -35 elements is accompanied by promoter bending and suggest that the major groove of the -10 hexamer widens to accommodate sigma  (33). Such a promoter structure would allow favorable interactions between basic residues Arg-441 and Arg-446 and the phosphate backbone (T-A base steps of the -10 promoter consensus element distort double-strand DNA in solution) (34). The model in Fig. 7a places the carboxyl group of amino acid 448 1.0 nm from the amino group of residue 454. This would allow for a flexible unstructured 7-amino acid loop connecting the two helices shown. Methods of probing for single-stranded DNA assign position -12 as the upstream limit of the open complex (35, 36). The TG motif at position -14/15 would thus remain in a region of double-stranded DNA, whereas helix 14 is shown within the transcription bubble (Fig. 7b).

    RESULTS

Construction and Conjugation of sigma 70 Mutants with FeBABE-- In previous work, we used suppression genetics to identify a region of the sigma 70 subunit of RNA polymerase, region 2.5, that interacts with the extended -10 motif of bacterial promoters (12). In this work, we have exploited a tethered DNA-cleaving agent to show that region 2.5 of sigma 70 is in close proximity to the extended -10 motif. The reagent used was FeBABE, which is covalently attached to sigma 70 by conjugation with a cysteine residue. Starting with an rpoD gene that had been mutated to remove all three native cysteine codons, single cysteine codons were introduced at amino acid positions 422, 454, 458, 459, 461, and 581. Residue 422 lies within region 2.3, adjacent to region 2.4, which is known to contact the -10 hexamer. Residues 454, 458, 459, and 461 are within region 2.5, postulated to contact the extended -10 motif, and residue 581 lies in region 4.2, which is known to contact the -35 hexamer element. The FeBABE reagent was conjugated to the single cysteine proteins, and the efficiency of conjugation was determined. The FeBABE conjugation yields were: 422C, 46%; 454C, 82%; 458C, 59%, 459C, 60%; 461C, 56%; 581C, 71%.

In this study we analyzed the interaction of E. coli RNA polymerase-carrying FeBABE with different promoters. We chose promoters that were sufficiently strong such that, in our conditions, open complex formation would not be disrupted by the introduction of the bulky FeBABE probe into region 2.5 of sigma 70. Thus, Figs. 2 and 3 show the transcription activity of RNAP holoenzyme preparations containing single-cysteine derivatives of sigma 70 before and after conjugation with FeBABE. With the exception of 422C, all derivatives retained at least 80% activity compared with wild-type before conjugation and at least 70% activity after conjugation with FeBABE.


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Fig. 2.   Transcriptional activity of RNAP carrying sigma 70 derivatives. The figure shows run-off transcripts from promoters (i) KAB-TG and (ii) KAB-TTcon. The single cysteine derivative is indicated. Transcripts generated by RNAP containing sigma 70 tagged with FeBABE are indicated by a +.


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Fig. 3.   Quantification of the transcription activity of RNAP carrying sigma 70 derivatives. The activity of the sigma 70 derivatives relative to wild-type sigma 70 (100%) at KAB-TTcon (a) and KAB-TG (b). The black bars represent activity of derivatives before conjugation, and the gray bars show activity after FeBABE conjugation and correction for the presence of unconjugated sigma 70.

Specificity of FeBABE Cleavage-- In our first experiment, we examined the cleavage of a labeled PstI-HindIII fragment purified from pAA121 carrying the KAB-TG promoter (Fig. 1), using RNAP containing sigma 70 tagged with FeBABE positioned at 461. This DNA fragment contains the KAB-TG promoter as well as the pbla and pX (these promoters are located upstream from the EcoRI site in the pAA121 vector). The results in Fig. 4 show that cleavage is observed with holoenzyme reconstituted from FeBABE-conjugated 461C mutant sigma . In contrast, no cleavage was observed with holoenzyme containing either wild-type sigma 70 or unconjugated 461C. The results (Fig. 4, lane 4) clearly show that cleavage is restricted to the three promoters. Further analysis revealed that similar patterns of FeBABE-mediated cleavage are observed at the KAB-TG, pbla, and pX promoters (data not shown).


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Fig. 4.   DNA cleavage pattern of a promoter fragment by RNAP carrying FeBABE attached at position 461 of sigma 70. The fragment used contained the KAB-TG, pbla, and pX promoters. Lane 1, no protein; lane 2, sequence calibration; lane 3, core RNAP; lane 4, RNAP containing the 461C derivative of sigma 70; lane 5, RNAP carrying FeBABE at Cys-461. The three promoters KAB-TG, pbla, and pX are indicated.

In our second set of experiments, both strands of the consensus extended -10 promoter galP1(19T8A9A) and the semi-synthetic promoters KAB-TG and KAB-TTcon (Fig. 1) were analyzed for cleavage by holoenzymes carrying sigma 70 protein tagged with FeBABE at different positions. The first promoter, galP1(19T8A9A), is a derivative of galP1, which has been changed to introduce a consensus -10 element. It is an extended -10 promoter containing a UP element but has a -35 hexamer with no homology with the consensus (25). To investigate the effects of a -10 extension on the pattern of cleavage, we also compared the KAB-TG and KAB-TTcon promoters. These promoters, which are also derived from the galP1 sequence, have similar activities. KAB-TG carries an extended -10 motif and canonical -10 (4/6 fit to consensus) and -35 (5/6 fit to consensus) hexamer sequences. KAB-TTcon lacks the extended -10 motif but carries an improved -35 (6/6 fit to consensus) hexamer.

Nontemplate Strand Cleavage-- Fig. 5a shows the patterns of cleavage on the nontemplate strand of the galP1(19T8A9A), KAB-TG, and KAB-TTcon promoters in open complexes with RNAP holoenzymes carrying sigma 70 tagged with FeBABE at positions 581, 422, 454, 458, 459, and 461. For each holoenzyme, the pattern of cleavage is similar at all three promoters, with some small variations in the positions and intensities of cleavage. For example, some upstream cleavages at the galP1 (19T8A9A) promoter are reduced. This is consistent with this promoter's lack of homology to the consensus within the -35 hexamer, leading to weaker interactions with upstream sequences. However, the overall similarity in cleavage patterns suggests that the organization of the different parts of sigma 70 is the same, irrespective of the precise promoter sequence. In contrast, changes in the position of FeBABE in sigma 70 result in marked variations in the positions of cleavage. Cleavage by FeBABE tethered at positions 581 (region 4.2) and 422 (region 2.3) of sigma 70 have been studied previously in open complexes at the lacUV5 promoter (37). The data here are in agreement with those previously published. FeBABE attached to 581C cleaved promoter DNA at positions -44/-45, -34 to -37, and -24 to -26. FeBABE positioned on 422C cleaved promoter DNA with very low efficiency at position -13/-12. The FeBABE positioned within region 2.5 (at residues 454, 458, 459, and 461) resulted in cleavage around -20 for all the sigma 70 derivatives, with additional cleavages at other positions being dependent on which sigma 70 derivative was studied. The predominant position of cleavage resulting from FeBABE tethered at 458 is ~-20 for all three promoters; however, additional upstream cleavage around -36 is seen for complexes at the KAB-TG promoter. Cleavage by FeBABE attached to 459C is limited to DNA around position -20. FeBABE located at 454 cleaves the nontemplate strand at position -20 but also at -17/-16. Cleavage at -17/-16 is not observed for other sigma 70 derivatives. FeBABE positioned at 461 again cleaves at -20, but additionally, there is cleavage from -13 to -1.


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Fig. 5.   Detailed FeBABE-mediated DNA cleavage at three promoters nontemplate strand (a) and template strand (b). The positions of FeBABE attachment to sigma 70 are indicated. The promoters studied are: 1, KAB-TTcon; 2, KAB-TG; 3, galP1(19T8A9A); M, Maxam-Gilbert G+A tracks (shown as calibration markers).

Template Strand Cleavage-- Fig. 5b shows cleavage on the template strand of the different promoters by FeBABE, located at different positions in sigma 70. Differences in the positions and intensities of DNA cleavage were observed compared with the nontemplate strand. A single region of promoter DNA at positions -13 and -14 is sensitive to hydroxyl radical attack by FeBABE when attached to all four positions in region 2.5. In addition FeBABE tethered at 458 and 459 cleaves at positions -21/-22, and 458C generates more distal cleavage at -38 to -40. FeBABE at 454C also gives unique cleavage at -18/-19 and, in common with 461C, cleaves downstream at -7/-8. There is increased cleavage at -7/-8 by FeBABE positioned at 461. Faint upstream cleavage for FeBABE attached at 461 was seen at -21/22 (common to 458C and 459C). Promoter-specific differences were seen in the cleavage pattern from FeBABE at 454, with an increase in the intensity of bands at -8 corresponding to an alteration of promoter bias toward the -10 element. As previously observed, FeBABE positioned at 581 in conserved region 4.2 cleaves promoter DNA from -38 to -41 and at -28/-29, whereas 422C in conserved region 2.3 gives weak cleavage at -16/-17. Fig. 6 shows the cleavage pattern from both strands of promoter KAB-TG in schematic form.


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Fig. 6.   Summary of cleavage pattern at KAB-TG. Boxes above each horizontal line represent nontemplate strand cleavage. Boxes below the line indicate template strand DNA cleavage. The "gray scale" reflects the observed cleavage efficiency of DNA, black being more efficient. The position of single cysteine replacement in sigma 70 for FeBABE tagging is shown next to each line. Numbers below the tick marks provide a reference for the positions of strand cleavage along the DNA template.


    DISCUSSION

This study and previous work (37) has shown that the cysteine residues present in the wild-type sigma 70 subunit of RNA polymerase are not essential for transcription initiation. Thus, it is possible to introduce cysteine residues in sigma 70 and to attach the DNA cleavage agent FeBABE and still retain transcriptional competence. In this work, FeBABE attached to the sigma 70 subunit of RNA polymerase was used to probe open complexes at a set of related promoters. The first important point to note is that the overall structure of RNA polymerase bound at promoters carrying different sequence elements appears to be very similar; the cleavage data was similar for all three variants of the galP1 promoter (Fig. 1) as well as the pX and pbla promoters. FeBABE attached to positions 422 in region 2.3 and 581 in region 4.2 were used as controls in our experiments, and the data are similar to those observed in a previous study (37). This is consistent with the view that RNA polymerase uses a variety of protein-DNA interactions to form essentially the same open complex at different classes of promoters. Note that the principal information gained from this technique concerns location, and the relative intensities of different bands cannot be interpreted to give detailed information about binding mechanisms. For this reason, we chose to work with strong promoters, where open complex formation would not be hindered by the bulky substitution of FeBABE.

The major conclusion from this study is that, in open complexes, region 2.5 of sigma 70 is close to promoter DNA sequences just upstream of the -10 hexamer. One aim of this work was to propose a model for the interaction of region 2.5 of sigma 70 with promoter DNA. Interpretation of the data is complicated by the fact that the holoenzymes carrying FeBABE-modified sigma 70 proteins are interacting with DNA that is known to be both bent and unwound. However, Fig. 7 shows possible models of how regions 2.4 and 2.5 of sigma 70 may interact with promoter DNA, based on the data obtained from this study and previous genetic work as discussed below. The similarity of cleavage pattern observed for FeBABE tethered at 454 and 461 and for FeBABE tethered at 458 and 459 is consistent with region 2.5 being alpha -helical. The cleavage pattern seen for FeBABE at 459 indicates that the reagent contacts the double-stranded promoter DNA around position -20. In the model, consistent with the length of the FeBABE spacer arm, the beta  carbon of Thr-459 is approximately 1.2 nm away from the proposed position of radical release. The FeBABE attached to 459C may be constrained, allowing it to sit in only one position relative to the DNA. We propose that position 459 is buried by the helix of region 2.5. FeBABE positioned at 458 results in the same cleavage pattern seen for 459C, but with additional weaker upstream DNA cleavage around the -35 hexamer, which may be because of bending and wrapping of upstream DNA sequences (33). The cleavage patterns observed with FeBABE positioned at 454 and 461 are more complex. We suggest that this complexity arises from the location of these side chains in an exposed position in the region 2.5 helix and the fact that promoter DNA is melted downstream from position -12 (12, 38).


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Fig. 7.   Modeling of possible interactions between promoter DNA and sigma 70 subunit of RNA polymerase. a shows the possible orientation of helix 14 with respect to the -10 element of promoter DNA. Residues thought to be important for promoter melting, Tyr-430, Trp-433, and Trp-434 are shown in purple. The distance of these residues from the site of nucleation of melting is shown by dotted lines. Residues Gln-437 (blue) and Thr-440 (green) are shown within hydrogen-bonding distance (dashed line) of the base edge of the thymine residue at position -12 of the nontemplate strand. Basic residues Arg-446 and Arg-441, which may interact with the negatively charged phosphate backbone of DNA, are shown in light blue. A schematic representation of the alpha -carbon backbone of region 2.5 of sigma 70 (454-461) shows a alpha -helix docked in the major groove with Glu-458 (red) in hydrogen-bonding contact with the base edges of adjacent template strand adenine (-15) and cytosine (-14). b shows a graphical interpretation of the cleavage pattern observed by attaching FeBABE to cysteine residues within region 2.5 of sigma 70. This space-filling model of region 2.5 shows wild-type amino acid side chain positions that were substituted by cysteine for FeBABE conjugation: Val-454 (gold), Thr-459 (green), and Asn-461 (red). Glu-458 (blue) is buried and not visible. On the basis of the cleavage patterns observed, the proposed locations of free radical generation (i.e. the position of the iron within FeBABE) are shown as star bursts. Starbursts are colored to reflect their origins and are placed 1.2 nm from the beta -carbon of the respective amino acid side chain. The diffusion path of hydroxyl radicals is indicated by arrows, with the efficiency of cleavage reflected by the thickness of the line. More distal DNA cleavage around the -35 hexamer by FeBABE tagged to a cysteine side chain at position 458 are not shown but are referred to by two horizontal blue arrows. Helix 14 (29) is shown schematically in close contact with the nontemplate strand, behind the template strand. The suggested arrangement of unwound promoter DNA provides an explanation for the observed cleavage pattern.

The model presented in Fig. 7b shows a conformation for open complex promoter DNA consistent with the cleavages observed from FeBABE at positions 454, 458, and 459, and 461. The modeling of region 2.5 as an alpha -helix is consistent with the data where FeBABE at positions 458 and 459 (100° apart on the alpha -helix) only cleave promoter DNA upstream on the TG motif, and FeBABE cleavages from positions 454 and 461 span the TG motif. The FeBABE cleavage upstream of the TG is best modeled with double-stranded DNA. In contrast, the complex pattern downstream of the TG motif cannot be modeled on double-stranded DNA, because the DNA was probed in the open complex. Modeling of region 2.5 as an alpha -helix in contact with the TG motif has important consequences for the orientation of sigma 70 and, in particular, region 2.4. Thus, in addition to the results presented here, we used previous genetic data to orientate both regions 2.4 and 2.5 with respect to the promoter (12, 30, 31). The aromatic residues Tyr-430 and Trp-433 are implicated in DNA melting and believed to interact at the -10/-11 positions on promoter DNA. The Gln-437 and Thr-440 residues interact with the base pair at -12. The Glu-458 residue is involved in the binding of the extended -10 motif at -14/-15 by region 2.5 of sigma 70. Hence the structures shown in Fig. 7, a and b, represent proposed orientations of regions 2.4 (helix 14) and 2.5 that can account for both the genetic and the biophysical data. Note that the orientation of helix 14 proposed in Fig. 7 is different to that suggested by Owens et al. (37) on the basis of cleavage patterns generated by FeBABE, located at positions 132, 376, 396, and 422 in sigma 70. Our present data is insufficient to prove either proposal (because of flexibility both in the DNA and in sigma 70 just downstream of helix 14). For example, increased DNA distortion could fit the data presented by Owens et al. to the models shown in Fig. 7. Similarly our data could be fitted to the Owens et al. model if a sharp kink is introduced into sigma 70 between helix 14 and region 2.5.

According to the model presented here (Fig. 7b), the seven amino acids immediately downstream of helix 14 form a junction between separate domains of sigma 70 (39). Flexibility of the loop would allow movement of region 2.4 relative to 2.5 during open complex formation. In the closed complex, this loop constrains the helix of region 2.4 relative to promoter DNA in the orientation shown (Fig. 7b). This model shows how region 2.5 can serve as an anchor, providing a scaffold on which the open complex may be built. These observations support the idea that the TG motif sets a limit on the conformational fluctuation of the -10 region (34, 40, 41). This is consistent with analysis of the temperature dependence of promoter opening at galP1 and supports a mechanism of open complex formation whereby melting nucleates around -10 (42, 43). Such a feature would be of particular importance at extended -10 promoters that lack an identifiable -35 hexamer.

    ACKNOWLEDGEMENTS

We are most grateful to the Wellcome Trust for generous funding of this work with a project grant and to the Royal Society for an Anglo-Japanese Scientific Collaboration Award. We thank Virgil Rhodius for help with the molecular modeling.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Tel.: +44-121-414-5438; Fax: +44-121-414-7366; E-mail: s.d.minchin{at}bham.ac.uk.

The abbreviations used are: RNAP, RNA polymerase; FeBABE, p-bromoacetamidobenzyl-EDTA·Fe.

2 http://www.pdb.bnl.gov.

    REFERENCES
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Abstract
Introduction
References

  1. Gross, C. A., Chan, C. L., and Lonetto, M. A. (1996) Philos. Trans. R. Soc. Lond-Biol. Sci. 351, 475-482[Medline] [Order article via Infotrieve]
  2. Gross, C. A., Lonetto, M., and Losick, R. (1992) in Transcriptional Regulation (McKnight, S. L., and Yamamoto, K. R., eds), pp. 129-176, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  3. Hawley, D. K., and McClure, W. R. (1983) Nucleic Acids Res. 11, 2237-2255[Abstract]
  4. Ross, W., Gosink, K. K., Salomon, J., Igarashi, K., Zou, C., Ishihama, A., Severinov, K., and Gourse, R. L. (1993) Science 262, 1407-1413[Medline] [Order article via Infotrieve]
  5. Bown, J. A., Barne, K. A., Minchin, S. D., and Busby, S. J. W. (1997) Nucleic Acids Mol. Biol. 11, 41-52
  6. Kumar, A., Malloch, R. A., Fujita, N., Smillie, D. A., Ishihama, A., and Hayward, R. S. (1993) J. Mol. Biol. 232, 406-418[CrossRef][Medline] [Order article via Infotrieve]
  7. Minchin, S. D., and Busby, S. J. W. (1993) Biochem. J. 289, 771-775[Medline] [Order article via Infotrieve]
  8. Helmann, J. D. (1995) Nucleic Acids Res. 23, 2351-2360[Abstract]
  9. Bashyam, M. D., and Tyagi, A. K. (1998) J. Bacteriol. 180, 2568-2573[Abstract/Free Full Text]
  10. Sabelnikov, A. G., Greenberg, B., and Lacks, S. A. (1998) J. Mol. Biol. 250, 144-155[CrossRef]
  11. Graves, M. C., and Rabinowitz, J. C. (1986) J. Biol. Chem. 261, 11409-11415[Abstract/Free Full Text]
  12. Barne, K. A., Bown, J. A., Busby, S. J. W., and Minchin, S. D. (1997) EMBO J. 16, 4034-4040[Abstract/Free Full Text]
  13. Heilek, G. M., Marusak, R., Meares, C. F., and Noller, H. F. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1113-1116[Abstract]
  14. Sundberg, M. W., Meares, C. F., Goodwin, D. A., and Diamanti, C. I. (1974) Nature 250, 587-588[Medline] [Order article via Infotrieve]
  15. Tullius, T. D., and Dombroski, B. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5469-5473[Abstract]
  16. Murakami, K., Kimura, M., Owens, J. T., Meares, C. F., and Ishihama, A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1709-1714[Abstract/Free Full Text]
  17. Murakami, K., Owens, J. T., Belyaeva, T. A., Meares, C. F., Busby, S. J. W., and Ishihama, A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11274-11278[Abstract/Free Full Text]
  18. Hanahan, D. (1983) J. Mol. Biol. 166, 557-580[Medline] [Order article via Infotrieve]
  19. Perrin, S., and Gilliland, G. (1990) Nucleic Acids Res. 18, 7433-7438[Abstract]
  20. Igarashi, K., and Ishihama, A. (1991) Cell 65, 1015-1022[Medline] [Order article via Infotrieve]
  21. Owens, J. T., Miyake, R., Murakami, K., Chmura, A. J., Fujita, N., Ishihama, A., and Meares, C. F. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 6021-6026[Abstract/Free Full Text]
  22. Barne, K. A. (1997) Isolation and Characterisation of an Altered Specificity Mutant of the Escherichia coli RNA Polymerase p70 Subunit. Ph.D thesis, The University of Birmingham, UK
  23. Bingham, A. H. A., Ponnambalam, S., Chan, B., and Busby, S. (1986) Gene 41, 67-74[CrossRef][Medline] [Order article via Infotrieve]
  24. Chan, B., and Busby, S. (1989) Gene 84, 227-236[CrossRef][Medline] [Order article via Infotrieve]
  25. Burns, H. D., Belyaeva, T. A., Busby, S. J. W., and Minchin, S. D. (1996) Biochem. J. 317, 305-311[Medline] [Order article via Infotrieve]
  26. Fujita, N., and Ishihama, A. (1996) Methods Enzymol. 273, 121-130[Medline] [Order article via Infotrieve]
  27. Greiner, D. P., Miyake, R., Moran, J. K., Jones, A. D., Negishi, T., Ishihama, A., and Meares, C. F. (1997) Bioconjugate Chem. 8, 44-48[CrossRef][Medline] [Order article via Infotrieve]
  28. Busby, S., Kolb, A., and Minchin, S. D. (1994) in Protein-DNA Interactions: Principles and Protocols (Neale, G. G., ed), Vol. 30, pp. 397-411, Humana Press Inc., Totowa, NJ
  29. Malhotra, A., Severinova, E., and Darst, S. A. (1996) Cell 87, 127-136[Medline] [Order article via Infotrieve]
  30. Siegele, D., Hu, J., Walter, W., and Gross, C. (1989) J. Mol. Biol. 206, 591-603[Medline] [Order article via Infotrieve]
  31. Waldburger, C., Gardella, T., Wong, R., and Susskind, M. M. (1990) J. Mol. Biol. 215, 267-276[Medline] [Order article via Infotrieve]
  32. deHaseth, P. L., and Helmann, J. D. (1995) Mol. Microbiol. 16, 817-824[Medline] [Order article via Infotrieve]
  33. Pérez-Martín, J., Rojo, F., and de Lorenzo, V. (1994) Microbiol. Rev. 58, 268-290[Abstract]
  34. Spassky, A., Rimsky, S., Buc, H., and Busby, S. (1988) EMBO J. 7, 1871-1879[Abstract]
  35. Sasse-Dwight, S., and Gralla, J. D. (1989) J. Biol. Chem. 264, 8074-8081[Abstract/Free Full Text]
  36. Buckle, M., and Buc, H. (1994) in Transcription: Mechanisms and Regulation (Conaway, R. C., and Conaway, J. W., eds), pp. 207-225, Raven Press, Ltd., New York
  37. Owens, J. T., Chmura, A. J., Murakami, K., Fujita, N., Ishihama, A., and Meares, C. (1998) Biochemistry 37, 7670-7675[CrossRef][Medline] [Order article via Infotrieve]
  38. Chan, B., Minchin, S., and Busby, S. (1990) FEBS Lett. 267, 46-50[CrossRef][Medline] [Order article via Infotrieve]
  39. Severinova, E., Severinov, K., Fenyö, D., Marr, M., Brody, E. N., Roberts, J. W., Chait, B. T., and Darst, S. A. (1996) J. Mol. Biol. 263, 637-647[CrossRef][Medline] [Order article via Infotrieve]
  40. Chen, Y. F., and Helmann, J. D. (1997) J. Mol. Biol. 267, 47-59[CrossRef][Medline] [Order article via Infotrieve]
  41. Juang, Y. L., and Helmann, J. D. (1995) Biochemistry 34, 8465-8473[Medline] [Order article via Infotrieve]
  42. Burns, H., and Minchin, S. (1994) Nucleic Acids Res. 22, 3840-3845[Abstract]
  43. Grimes, E., Busby, S., and Minchin, S. (1991) Nucleic Acids Res. 19, 6113-6118[Abstract]


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