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

2000; Springer Nature; Volume: 19; Issue: 12 Linguagem: Inglês

10.1093/emboj/19.12.3038

1460-2075

Sivaramesh Wigneshweraraj, Nobuyuki Fujita, Akira Ishihama, Martin Buck,

Genomics and Phylogenetic Studies

Article15 June 2000free access Conservation of sigma–core RNA polymerase proximity relationships between the enhancer-independent and enhancer-dependent sigma classes Siva R. Wigneshweraraj Siva R. Wigneshweraraj Imperial College of Science, Technology and Medicine, Department of Biology, Sir Alexander Fleming Building, Imperial College Road, London, SW7 2AZ UK Search for more papers by this author Nobuyuki Fujita Nobuyuki Fujita Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka, 411 Japan Search for more papers by this author Akira Ishihama Akira Ishihama Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka, 411 Japan Search for more papers by this author Martin Buck Corresponding Author Martin Buck Imperial College of Science, Technology and Medicine, Department of Biology, Sir Alexander Fleming Building, Imperial College Road, London, SW7 2AZ UK Search for more papers by this author Siva R. Wigneshweraraj Siva R. Wigneshweraraj Imperial College of Science, Technology and Medicine, Department of Biology, Sir Alexander Fleming Building, Imperial College Road, London, SW7 2AZ UK Search for more papers by this author Nobuyuki Fujita Nobuyuki Fujita Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka, 411 Japan Search for more papers by this author Akira Ishihama Akira Ishihama Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka, 411 Japan Search for more papers by this author Martin Buck Corresponding Author Martin Buck Imperial College of Science, Technology and Medicine, Department of Biology, Sir Alexander Fleming Building, Imperial College Road, London, SW7 2AZ UK Search for more papers by this author Author Information Siva R. Wigneshweraraj1, Nobuyuki Fujita2, Akira Ishihama2 and Martin Buck 1 1Imperial College of Science, Technology and Medicine, Department of Biology, Sir Alexander Fleming Building, Imperial College Road, London, SW7 2AZ UK 2Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka, 411 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:3038-3048https://doi.org/10.1093/emboj/19.12.3038 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Two distinct classes of RNA polymerase sigma factors (σ) exist in bacteria and are largely unrelated in primary amino acid sequence and their modes of transcription activation. Using tethered iron chelate (Fe-BABE) derivatives of the enhancer-dependent σ54, we mapped several sites of proximity to the β and β′ subunits of the core RNA polymerase. Remarkably, most sites localized to those previously identified as close to the enhancer-independent σ70 and σ38. This indicates a common use of sets of sequences in core for interacting with the two σ classes. Some sites chosen in σ54 for modification with Fe-BABE were positions, which when mutated, deregulate the σ54–holoenzyme and allow activator-independent initiation and holoenzyme isomerization. We infer that these sites in σ54 may be involved in interactions with the core that contribute to maintenance of alternative states of the holoenzyme needed for either the stable closed promoter complex conformation or the isomerized holoenzyme conformation associated with the open promoter complex. One site of σ54 proximity to the core is apparently not evident with σ70, and may represent a specialized interaction. Introduction The Escherichia coli RNA polymerase is a multisubunit enzyme that exists in two major forms with respect to its subunit composition. The core enzyme (α2ββ′; E) is capable of processive transcription elongation followed by termination, but is unable to initiate transcription specifically unless it is in its σ-bound holoenzyme form (Eσ). The σ subunit confers promoter-specific DNA binding and transcription initiation capabilities on the RNA polymerase. Based on structural and functional criteria, the seven different σ factors (σ70, σ54, σ38, σ32, σ28, σ24 and σ18) identified in E.coli are grouped into two classes. Most σ factors, with the exception of σ54, belong to the σ70 class, the major σ factor in E.coli that is involved in expression of most genes during exponential growth. σ54 (σN) represents a unique class that differs functionally from the σ70 class. The presence of σ54 has been well documented in several proteobacteria as well as in the Gram-positive Bacillus subtilis (Debarbouille et al., 1991) and in Planctomyces limnophilus (Leary et al., 1998). σ54 directs its holoenzyme to recognize and bind promoters with conserved −12/−24 regions located between positions −11 and −26 relative to the transcription start. These can be considered the functional analogue of the −10/−35 core promoter elements recognized by σ factors belonging to the σ70 class. In E.coli, both σ70, the major σ factor for growth-related genes, and σ54 are always present, but other minor σ70 class members are induced under certain stress conditions (Jishage et al., 1997). Although σ54 binds the same core RNA polymerase as the σ70 class members, each σ class confers different properties on the holoenzymes formed. Eσ54 causes transcription to assume several features reminiscent of higher eukaryotic mechanisms (Sasse-Dwight et al., 1990). In marked contrast to the σ70–holoenzyme, which often initiates transcription in the absence of transcription activators, the σ54–holoenzyme remains in a closed promoter complex in the absence of activator protein. Isomerization of this closed complex to a transcriptionally competent open complex depends upon the activity of an activator protein bound to a DNA sequence with enhancer-like properties coupled to γ–β bond nucleoside triphosphate hydrolysis by the activator protein (Weiss et al., 1991). The functional domain organization of σ54 is complex and appears to differ significantly from that of the members of the σ70 class. In particular, overall DNA binding by σ70 is modulated by N-terminal sequences, whereas N-terminal sequences in σ54 are required for activation. Extensive deletion and mutational analyses of σ54 have allowed functions to be assigned to different regions of the protein (Merrick et al., 1993; Wong et al., 1994; Cannon et al., 1995; Gallegos and Buck, 1999). The N-terminal region I of σ54 is regulatory and closely implicated in polymerase isomerization and DNA melting (Cannon et al., 1999; Gallegos et al., 1999; Guo et al., 1999). It plays a central role in mediating the response to activator proteins and in binding early melted DNA structures (Gallegos and Buck, 2000; W.Cannon, M.T.Gallegos and M.Buck, submitted), but is dispensable for core and overall DNA binding (Cannon et al., 1999). Region II is acidic and not conserved. Region III includes a major core RNA polymerase-binding determinant and sequences that directly contact DNA and that enhance σ54–DNA binding (Merrick and Chambers, 1992; Cannon et al., 1997; Gallegos and Buck, 1999) (Figure 1A). Emerging roles for region III appear to be in maintaining the closed promoter complex in a transcriptionally silent state and in generating polymerase isomerization upon activation (Chaney and Buck, 1999). Figure 1.(A) Schematic representation of Klebsiella pneumoniae σ54 domain organization. The locations of the native cysteine residues are indicated. (B) Sites where a single cysteine was introduced and conjugated with Fe-BABE. The arrow indicates that conjugation was performed under denaturing conditions. Download figure Download PowerPoint The core RNA polymerase-binding interface of σ54 comprises at least two functionally distinct sequences: a 95 amino acid residue sequence (120–215) within region III, which binds the core strongly, and a second sequence within region I that binds the core more weakly (Gallegos and Buck, 1999). Comparison of hydroxyl radical-mediated cleavage profiles of wild-type and region I-deleted (ΔR1σ54) proteins indicated that the overall solvent-exposed surface of σ54 is not greatly altered by deletion of region I (Casaz and Buck, 1999). Residues protected from hydroxyl radical-mediated cleavage in the holoenzyme made with ΔR1σ54 largely coincided with residues protected specifically by DNA in the wild-type holoenzyme, implying a role for region I in establishing an appropriate conformation of the DNA-binding domain in the wild-type holoenzyme (Casaz and Buck, 1999). Thus, it is possible that the interaction of region I with core RNA polymerase is important for controlling DNA-binding domain conformation. To date, little is known about the location of σ54-binding sites on the core RNA polymerase. Recently, we showed by small-angle X-ray scattering studies that core RNA polymerase-binding fragments of σ54 (amino acids 70–324) and the crystal structure of core RNA polymerase-binding σ70 fragment (amino acids 114–448) have a similar envelope shape, leading to the suggestion that both σ classes might be located in similar places on the core (Svergun et al., 2000). However, the sequence differences that exist between σ54 and σ70 must also account for the markedly different properties of the two holoenzymes. The affinities of σ54 and σ70 for the core RNA polymerase measured in vitro are similar (Gallegos and Buck, 1999). Both σ factors could use the same binding site on core RNA polymerase, have overlapping sites or utilize additional independent binding sites. Recent protein–protein interaction and footprinting studies have revealed that the β′ subunit of core RNA polymerase provides the major binding interaction for σ70 in the holoenzyme, while the β subunit contributes a further binding interaction (Arthur and Burgess, 1998; Owens et al., 1998; Katayama et al., 2000). In the present work, we have used tethered nucleophile-mediated footprinting to extend our understanding of the σ54–core RNA polymerase interface. Using (p-bromoacetamidobenzyl)-EDTA Fe (Fe-BABE) derivatives of σ54, we mapped several sites of proximity to the β and β′ subunits of the core RNA polymerase. Remarkably, most sites localized to those previously identified as close to enhancer-independent σ70 (Owens et al., 1998) and σ38 (A.Kolb, personal communication). This indicates use of the same or overlapping sequences in core RNA polymerase for interaction with the two σ classes. One site of σ54 proximity to the β′ subunit apparently is not evident with σ70, and may represent a specialized interaction of σ54 with core RNA polymerase. Results Location of Fe-BABE conjugation targets on σ54 The bifunctional chelating agent Fe-BABE covalently conjugates to the free sulfhydryl groups of cysteine residues. After the addition of ascorbate and peroxide, the Fe-BABE generates nucleophiles, which cleave polypeptide chains in proximity (∼12 Å) to the chelate, independently of the amino acid sequence involved (Rana and Meares, 1991; Ishihama, 2000). Using information from extensive mutational analysis of σ54, we selected five sites (E36, C198, R336, C346 and R383, described below) of potential interest for tethering Fe-BABE. Figure 1B shows the schematic representation of the primary structure of σ54, in which the positions of introduced cysteine residues are indicated. Cys198 and Cys346. The σ54 from Klebsiella pneumoniae contains two highly conserved cysteine residues at amino acid positions 198 and 346. Cys198 is located in a 95 amino acid sequence (120–215), which binds core RNA polymerase strongly and is likely to include most of the fold critical for forming stable holoenzyme (Gallegos and Buck, 1999). Cys346 borders a DNA-interacting patch in σ54 (Cannon et al., 1994) and is strongly protected by the core in hydroxyl radical cleavage experiments when region I is absent (Casaz and Buck, 1999). By changing Cys198 and Cys346 to alanine, we constructed two mutant σ54 proteins with single cysteines at positions 198 (C346A) and 346 (C198A). The Cys(−)rpoN construct was used to purify the cysteine-free double mutant (C198A and C346A) protein, which was used as the negative control protein in the cleavage assays. The Cys(−)σ54 protein was found to maintain 90% of its transcriptional activity (see later), implying that substitution of the conserved Cys198 and Cys346 with alanine does not significantly affect the conformation of the mutant protein. Cys36. In the next step, single cysteine residues were introduced into Cys(−)σ54. Region I of σ54 has multiple roles in regulating σ54 function: (i) inhibiting polymerase isomerization in the absence of activation and directing fork junction DNA binding (Wang et al., 1995; Syed and Gralla, 1998; Cannon et al., 1999; Guo et al., 1999); and (ii) stimulating initiation of open complex formation in response to activation (Sasse-Dwight and Gralla, 1990; Syed and Gralla, 1998; Casaz et al., 1999; Gallegos and Buck, 2000). The low affinity of region I sequences for core RNA polymerase and its protection from proteolysis by core (Casaz and Buck, 1997, 1999; Gallegos and Buck, 1999) prompted us to introduce a single cysteine residue into region I in the Cys(−)σ54 protein. The increased protease sensitivity of residue E36 detected upon open complex formation provided us with a potential target for introducing a cysteine that could be in close proximity to the core RNA polymerase subunits in closed complexes (Casaz and Buck, 1997). Cys336. Recently, we showed that a single amino acid substitution, R336A, in the region III DNA-binding domain of σ54 allows holoenzyme to isomerize and transcribe without activator (Chaney and Buck, 1999), a phenotype previously only associated with region I mutations (Hsieh and Gralla, 1994; Hsieh et al., 1994; Syed and Gralla, 1998; Casaz et al., 1999). We justify our choice of R336 for introducing a single cysteine residue by suggesting that R336 is a part of a network of interactions necessary for maintaining the transcriptionally silent state of the holoenzyme and thus might interact directly or indirectly with core RNA polymerase subunits. Cys383. The highly conserved arginine at position 383 is located within the second helix of the proposed helix–turn–helix motif in region III of σ54, suggested to make direct contact with the conserved −12 promoter sequences (Coppard and Merrick, 1991; Merrick and Chambers, 1992). Recent evidence shows conserved arginine residues in the region III DNA-binding domain of σ54 prevent polymerase isomerization (Chaney and Buck, 1999). Furthermore, the −12 promoter sequence is part of a molecular switch that has to be thrown by the action of the activator for transcription to proceed (Guo et al., 1999) through a network of communication possibly involving R383, other σ54 parts and/or the core subunits. We therefore targeted R383 to introduce a single cysteine residue. Single cysteine σ54 mutants are active for transcription Next, the activity of each single cysteine mutant and the Cys(−)σ54 protein was compared with the activity of wild-type σ54 in an in vitro activator-dependent single round transcription assay using the E.coli glnHp2-m12 promoter as the template (Claverie-Martin and Magasanik, 1992). Assays were conducted with subsaturating amounts of holoenzyme to allow quantitative detection of holoenzyme activities. The mutant proteins were found to exhibit >50% of the activity of wild-type σ54 (Figure 2A). We therefore conclude that a replacement of all endogenous cysteines with alanine residues and the introduction of cysteine residues at alternative positions (see above) had no gross negative effect on the function of the protein. Also, because transcriptional activity of σ54 involves numerous unique stereospecific interactions, the preservation of transcriptional activity in the single cysteine mutants and the Cys(−)σ54 protein suggests that the conformation of the mutant proteins important for function is not altered significantly. Figure 2.In vitro transcription activity of σ54 single cysteine mutants on supercoiled E.coli glnHp2-m12 promoter. (A) Activator-dependent (using PspFΔHTH) and (B) activator-independent transcription activity of σ54 single cysteine mutants and Cys(−)σ54 relative to the wild type. Δ(21–27)σ54 (Gallegos and Buck, 2000) was used as the positive control (Positive) in the activator-independent assays. Download figure Download PowerPoint Region I sequences and R336 are involved in preventing unregulated polymerase isomerization (Syed et al., 1997; Syed and Gralla, 1998; Casaz et al., 1999; Chaney and Buck, 1999). We therefore tested whether the single cysteine σ54 mutants were active for unregulated transcription (also called 'activator bypass' transcription), i.e. whether they allowed polymerase isomerization in the absence of activator proteins. The protocol for these assays begins by incubating the holoenzyme with GTP, CTP and UTP. The first six bases transcribed from the E.coli glnHp2-m12 template are UGUCAC (+1 to +6), so transcripts can be initiated under these conditions if an activator-independent open complex forms. ATP and [α-32P]UTP are then added, together with heparin, to destroy residual closed complexes and unstable open complexes, and allow elongation of any initiated transcripts. E36C and R336C showed activator-independent transcription activity (Figure 2B). The lack of unregulated transcription activity from the Cys(−)σ54 protein suggests that the bypass phenotype of E36C and R336C is attributable to the substitutions at amino acid residues at positions E36 and R336, and not because of the replacement of the two endogenous cysteines at positions C198 and C346. Consistent with previous observations by us (Casaz et al., 1999; Chaney and Buck, 1999) and others (Hsieh and Gralla, 1994; Hsieh et al., 1994; Syed and Gralla, 1998), E36 and R336 in σ54 have a function in inhibiting polymerase isomerization in the absence of activation. Derivatization of single cysteine σ54 mutants with the chemical protease Fe-BABE Each single cysteine σ54 mutant and Cys(−)σ54 were derivatized with the chemical cleavage reagent Fe-BABE under native conditions and the conjugation yield was determined fluorometrically by the CPM test (N-[4-[7-(diethylamino)-4-methylcoumarin-3-yl]phenyl]maleimide) (Greiner et al., 1997) from the fluorescence difference between the conjugated and unconjugated proteins. The conjugation yield for the single cysteine mutants E36C, R336C, C198A and R383C was estimated to be >40% (Table I). The mutant σ54 harbouring the native single cysteine at position C198 (the C346A mutation) proved difficult to conjugate under native conditions (conjugation yield of 20%), implying that C198 is probably slightly buried or located in a hydrophobic region in the folded state because the hydrophobic reagent CPM reacted with high yield (data not shown) while the hydrophilic molecule Fe-BABE did not. When conjugation with C198 was performed under denaturing conditions, the yield increased to 50%. The Cys(−)σ54 protein failed to react detectably with CPM and Fe-BABE under native and denaturing conditions, directly implying that under the conditions used, Fe-BABE conjugation has only occurred at the free sulfhydryl side groups of cysteine residues. Table 1. Fe-BABE conjugation efficiency Residue Mutation Conjugation efficiency (%) 36 E36C 53 198 C346A 46a 336 R336C 51 346 C198A 47 383 R383C 76 Cys(−) C198A/C346A 3/5a a Denotes conjugation under denaturing conditions. The Fe-BABE-derivatized single cysteine mutants of σ54 are active for holoenzyme formation and transcription We investigated the properties of the derivatized single cysteine mutants of σ54 proteins in vitro to determine whether core RNA polymerase binding and transcription were affected. Native gel holoenzyme assembly assays were used to detect complex formation between core RNA polymerase and σ54. The holoenzyme migrates more slowly than the core enzyme. With the exception of the conjugated R336C mutant, all derivatized σ54 mutant proteins bound core RNA polymerase with the same apparent affinity as the non-conjugated wild-type σ54 (Figure 3A). Residue 336 lies outside the minimal core-binding region defined by deletion mutagenesis (Gallegos and Buck, 1999), but adjacent to a region in σ54 (amino acids 290–310) that is protected by core RNA polymerase from hydroxyl radical-mediated cleavage (Casaz and Buck, 1999) and within a sequence (amino acids 325–440) strongly protected by core RNA polymerase when region I is removed (Casaz and Buck, 1999). Because the unconjugated R336C binds core RNA polymerase with an affinity similar to that of wild-type σ54, we conclude that the presence of the 490 Da Fe-BABE molecule at position R336 has caused the R336C mutant to undergo a conformational change that diminishes binding to the core RNA polymerase. We also note that conjugated Cys198 binding to core RNA polymerase results in two different holoenzyme conformers, one of which has a slower native gel mobility (Figure 3A, lane 10, marked with an arrow). Figure 3.(A) Formation of holoenzyme by the unconjugated (−) and Fe-BABE-conjugated (+) σ54 single cysteine mutants. Migration positions of free core RNA polymerase, holoenzyme and free σ protein are indicated. (B) In vitro transcription activity as in Figure 2. The activities of the σ54 single cysteine mutants before (black bars) and after (white bars) Fe-BABE conjugation are compared. The values for conjugated proteins are corrected for the presence of unconjugated proteins. (C) Activator bypass activity of the E36C and R336C σ54 proteins after Fe-BABE conjugation (C); the activator bypass activity of unconjugated proteins (UC) is shown for comparison. Download figure Download PowerPoint To determine the effect of Fe-BABE conjugation on the transcriptional activity of each single cysteine σ54 mutant, single-round transcription assays were also performed after conjugation with the Fe-BABE probe. Assays were conducted with subsaturating amounts of holoenzyme to allow detection of activity differences. After correction for the presence of unconjugated σ54 proteins, the conjugates showed >40% transcription activity relative to wild-type σ54, with the exception of the R336C mutant, which has a markedly reduced transcription activity (10%) following conjugation (Figure 3B), consistent with our previous observation that the conjugated R336C mutant has reduced affinity for the core. Nevertheless, the conjugated R336C σ54 forms a holoenzyme that is active for a significant level (10%) of activated transcription. We used the activator bypass transcription assay on the conjugated E36C and R336C mutants as a further indicator of conformational preservation of these proteins following conjugation. We reasoned that if the E36C and R336C mutants retained bypass activity following Fe-BABE conjugation, then the presence of the Fe-BABE molecule at these positions did not greatly affect the conformation of the conjugated proteins. Both mutants E36C and R336C retained their bypass activity following conjugation with Fe-BABE (Figure 3C). In conclusion, the core RNA polymerase-binding assays, the activator-dependent and the bypass transcription assays altogether suggest that Fe-BABE conjugation did not grossly affect the overall conformation and structural integrity of the mutant σ54 proteins. Enhancer-dependent σ54- and enhancer-independent σ70-interacting sites on β and β′ core subunits are conserved Holoenzymes were formed with conjugated and unconjugated single cysteine mutants of σ54 and cleavage reactions were carried out essentially as previously described (Owens et al., 1998). In our approach, we assigned the cleavage sites on the β and β′ subunits by comparing the relative migration of the Fe-BABE cleavage products with sequence-specific markers generated by cleaving β and β′ subunits at either cysteine or methionine residues [using 2-nitro-5-thiocyanobenzoate (NTCB) and cyanogen bromide (CNBr), respectively]. The cleavage sites for each β and β′ marker fragment were assigned by calculating the molecular weight of each fragment with respect to the locations of cysteine or methionine residues in the amino acid sequence of β and β′ subunits (Figure 4A) using a plot of log molecular weight versus the relative migration distance of a known set of marker proteins (data not shown). Figure 4.(A) Immunoblot of markers generated by treating core RNA polymerase with NTCB (N) and CNBr (C), respectively, using anti-β or anti-β′ N-terminus-specific antibodies. Pre-stained molecular weight markers (M; Bio-Rad, Low Range SDS–PAGE Standards) were used as reference molecular weight markers. Holoenzyme cutting by σ54 single cysteine–Fe-BABE conjugates. Immunostained blots of SDS–PAGE detecting either (B) β or (C) β′ subunit fragments by using affinity-purified N-terminal antibodies. All reactions were either treated (+) or untreated (−) with ascorbate and hydrogen peroxide. Download figure Download PowerPoint Initially, we showed that self-cleavage of σ54 did not occur (data not shown), indicating that cutting of β and β′ was attributed to binding of intact σ54 proteins. Ascorbate- and hydrogen peroxide-treated and untreated reactions of each single cysteine mutant σ54–holoenyzme were separated, blotted and visualized by immunostaining with affinity-purified β and β′ N-terminus-specific antibodies (Figure 4B and C). The lack of cleavage of free core RNA polymerase and the holoenzyme formed with Cys(−)σ54 (Figure 4B and C, controls) protein serves as an essential control experiment, demonstrating that under the conditions used there is no artefactual cutting of the core RNA polymerase subunits. Hence, we believe that each cut (Figure 4B and C) is attributed to a specific Fe-BABE form of σ54 created by derivatizing a single cysteine residue. Cys198 and Cys346. Initially, we looked for the β and β′ interactions made by the σ54 single cysteine mutants with Fe-BABE conjugated to the endogenous cysteines at positions Cys198 (C346A) and Cys346 (C198A), respectively. Cys198 cleaves β and β′ at multiple sites. On the β subunit, strong cleavage by Cys198 is seen within the Rif1 cluster (between conserved β regions C and D) and in conserved region G (Figure 4B, Cys198, lane 4). A few weak cleavages are also seen within the DR1 region. Moderate cleavage by Cys198 of the β′ subunit occurs in conserved regions B and C and within region G (Figure 4C, Cys198, lane 2). In contrast, Cys346 only moderately cleaves β and β′ at single sites; these map proximal to the conserved region D on the β subunit and within conserved regions C and D on the β′ subunit (Cys346, lane 6, Figure 4B and C, respectively). These results are consistent with previous observations: Cys198 is located within a sequence (amino acids 120–215) in region III of σ54 that has a strong affinity for the core RNA polymerase (Gallegos and Buck, 1999), while Cys346 is located within a region (amino acids 325–340) that is weakly protected by core RNA polymerase during hydroxyl radical cleavage (Casaz and Buck, 1999), consistent with the differences in the β and β′ cleavage patterns generated by the Cys198- and Cys346-conjugated σ54 proteins. Cys 36 and Cys336. Next, we used the activator-independent single cysteine mutants of σ54 with Fe-BABE conjugated to Cys36 (E36C) and Cys336 (R336C) in the Cys(−)σ54 background to map their interaction sites on the β and β′ subunits of core RNA polymerase. Cys36 is in region I of σ54, which functions to keep the closed complexes in a transcriptionally silent state in the absence of activation (Gallegos et al., 1999) and has been shown to have weak affinity for core RNA polymerase (Gallegos and Buck, 1999). The core RNA polymerase cleavage by Cys36 shows that region I interacts strongly with β and β′ subunits. Cleavage sites map to within the Rif1 cluster and the conserved region D on β and within conserved region C on β′ (Cys36, lane 2, Figure 4B and C). Cys336 is located within a sequence in σ54 that lies outside the minimal DNA-binding domain, but nevertheless cross-links to DNA (Chaney and Buck, 1999; M.K.Chaney, M.S.Pitt and M.Buck, submitted). Remarkably, the cleavage data indicate that Cys336 interaction sites on both β and β′ subunits unambiguously overlap with those of Cys36. Given the shared activator-independent transcription phenotypes of the E36C and R336C σ54 mutants, we rationalize this observation by suggesting that E36 and R336 belong to an interface between σ54 and core RNA polymerase that acts to prevent polymerase isomerization in the absence of activators, and propose that the activator probably functions in changing the interface. Cys383. Attempts to cleave core RNA polymerase by Cys383 (R383C)-conjugated σ54 revealed no discernible cleavage of either the β or β′ subunit (Cys383, lane 10, Figure 4B and C, respectively). In σ54, Cys383 is proposed to fall within the recognition helix (helix 2) of the putative helix–turn–helix motif and to contribute to the recognition of the −12 promoter element by the σ54–holoenzyme (Coppard and Merrick, 1991; Merrick and Chambers, 1992). We can conclude that Cys383 is not located proximal (at least not within a radius of 12 Å) to the core RNA polymerase subunits β and β′ and may be facing promoter DNA rather than core. A comparison with σ70–core RNA polymerase proximity. Because binding of σ54 to core RNA polymerase results in the formation of a holoenzyme that has very distinct functional properties compared with the σ70–holoenzyme, we compared our cleavage data with those obtained for σ70–holoenzyme cleavage by Fe-BABE-