Структурный анализ признания35 Пропагандистский элемент SigW из Bacillus subtilis.

Introduction

Transcription in bacteria is initiated by sigma factors, which recruit the core RNA polymerase to a cognate promoter [ 1 , 2 ]. Sigma factors selectively recognize promoter elements, -10 and -35 elements, and additional sequences, including extended -10 element and discriminator, which are present upstream of the transcription start site [ 3 , 4 ]. During transcription initiation, the -10 element is strand-separated to form a transcription bubble [ 5 , 6 ], whereas the -35 element is recognized by a helix-turn-helix (HTH) motif in the sigma factor, without strand separation [ 7 , 8 ].

Sigma factors are categorized into two families, based on the sequence homology with Escherichia coli sigma factors: housekeeping σ 70 required for bacterial homeostasis; and σ 54 activated for nitrogen utilization [ 9 ]. The σ 70 family is further sub-divided into five groups [ 4 , 10 ]. Group I sigma factors are composed of σ 1.1 , σ 2 , σ 3 , and σ 4 domains, which are responsible for the recognition of the discriminator, -10, extended -10, and -35 elements, respectively. The primary sigma factors, which regulate the transcription of housekeeping genes, belong to group I. Group II-V are classified depending on the presence or absence of the domains and motifs in group I. Alternative sigma factors, which are activated in response to a range of stress conditions, belong to groups II-V [ 10 ].

Group IV sigma factors are known as extracytoplasmic function (ECF) sigma factors, because they are activated in response to extracytoplasmic stresses [ 3 , 10 , 11 ]. They are the most divergent group of sigma factors, and almost all bacteria contain multiple ECF sigma factors. As an extreme case, Streptomyces coelicolor contains approximately 50 ECF sigma factors in its genome [ 10 , 12 ]. The ECF sigma factors are composed of only σ 2 and σ 4 domains, which bind -10 and -35 elements, respectively [ 3 , 13 – 15 ]. In many cases, promoter binding of ECF sigma factors is inhibited by binding to the cytoplasmic domain of transmembrane anti-sigma factors [ 10 , 11 , 16 ], and is activated to initiate the transcription of target genes in response to a specific stress signal by being released from the anti-sigma factor.

Bacillus subtilis contains at least seven ECF sigma factors: SigM, SigV, SigW, SigX, SigY, SigZ, and YlaC [ 17 ]. Of these sigma factors, SigW induces the transcription of its target regulon to counteract cell envelope stresses caused by antibiotics [ 18 , 19 ], alkaline pH [ 20 ], and high salt concentration [ 21 , 22 ]. In the absence of these stresses, SigW is downregulated by anti-sigma factor RsiW, which is localized in the plasma membrane [ 16 , 23 ]. It is released from the anti-sigma factor under the stress conditions [ 24 – 27 ]. Under these conditions, transmembrane RsiW is sequentially cleaved by regulated intramembrane proteolysis by PrsW and RasP proteases; therefore, the cytoplasmic domain of RsiW is released together with SigW from the plasma membrane [ 25 , 27 ]. Subsequently, the cytoplasmic domain of RsiW is completely degraded by ClpXP protease [ 28 ], allowing SigW to bind to the core RNA polymerase, and thereby to activate SigW-dependent transcription.

Even though crystal structures have been reported showing interactions between the σ 4 domain and the -35 promoter element in group I and group IV sigma factors [ 3 , 7 , 8 , 29 , 30 ], structural information from diverse sigma factors is required to understand the selective recognition of the -35 element. In the work reported here, we determined the crystal structure of the σ W 4 /-35 element complex, and analyzed the promoter binding mode of SigW. The structure reveals the unique regulation mode of SigW-dependent transcription, and the recognition specificity of the -35 element by the σ 4 domain.

Materials and methods

Plasmid preparation and protein expression

DNA encoding the σ4 domain of B . subtilis SigW (σ W 4 ; residues 126–187) was amplified from the genome of B . subtilis 168 strain using polymerase chain reaction and inserted into pETDuet-1 vector (Merck Millipore, Billerica, MA, USA) expressing the N-terminal 6 X His tag and TEV protease cleavage site. The plasmid was transformed into E . coli strain BL21-star (DE3) (Thermo Fischer Scientific, Waltham, MA, USA) and cells were grown in Luria Broth media at 37°C. When the culture reached an OD 600 of 0.6–0.7, the medium was cooled, and 0.4 mM isopropyl β-D-1-thiogalactopyranoside was added to the culture to induce σ W 4 expression. After overnight incubation at 15°C, the cells were harvested by centrifugation at 3,000 g for 10 min.

Purification of σ W 4 /-35 W

Cells expressing 6 X His-σ W 4 were resuspended in buffer A (20 mM HEPES pH 7.5, 1.0 M NaCl, and 10% (v/v) glycerol), lysed by sonication, and clarified by centrifugation at 20,000 g for 30 min, after the addition of DNase I and RNase A at a concentration of 10 μg/ml. σ W 4 was purified by immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Clarified cell lysate was loaded onto a 5 mL HisTrap nickel chelating column (GE Healthcare Bio-sciences, Uppsala, Sweden), and the resin was washed with buffer A, containing 80 mM imidazole. Proteins bound to the resin were eluted by an imidazole gradient (0.08–1.00 M imidazole). Fractions that contained 6 X His-σ W 4 were pooled and treated with TEV protease overnight at 25°C to cleave the His tag. After complete cleavage, the protein solution was dialyzed against buffer A for 3 h and passed through Ni-NTA resin to remove the 6 X His tag (Thermo Fisher Scientific, Rockford, IL, USA). σ W 4 was further purified by SEC using Superdex 75 preparatory grade column (GE Healthcare Biosciences) pre-equilibrated with buffer B (20 mM HEPES pH 7.5, 1.0 M NaCl, and 5% (v/v) glycerol).

The -35 element recognized by σ W 4 was prepared by mixing two complementary DNA fragments. Two single-stranded DNAs (5’-ATTGAAACCTTT-3’ and 5’-AAAAGGTTTCAA-3’) were synthesized (Biobasic, Seoul, South Korea), mixed at a 1:1 molar ratio in buffer B, and purified by SEC using a Superdex75 analytical column. The purified -35 element (-35 W ) was then mixed with σ W 4 at a 1.1:1 molar ratio. The mixture was dialyzed in buffer C (20 mM HEPES pH 7.5, 0.2 M NaCl, and 5% (v/v) glycerol) and concentrated to 15 mg/mL for crystal screening.

Crystallization, data collection, and structure determination

Crystallization of the σ W 4 /-35 W complex was performed using the micro-batch method at 20°C. The drop for crystal screening was prepared by mixing 1 μL of σ W 4 /-35 W (15 mg/mL) and 1 μL of crystallization solution under a layer of Al’s oil (Hampton Research, Aliso Viejo, Ca, USA). Crystals of σ W 4 /-35 W grew completely in a month under the conditions of Wizard Precipitant Synergy 127 (0.1M imidazole/hydrochloric acid pH 6.5, 30% (v/v) PEG1500, 10% (v/v) isopropanol, and 0.1 M CaCl 2 ) (Rigaku, Tokyo, Japan). Crystals of σ W 4 /-35 W were picked using a cryo-loop (Hampton Research) and flash-frozen in a cold nitrogen stream. Diffraction data were collected at PLS-BL7A (Beam line 7A, Pohang Light Source, South Korea) [ 31 ] and were indexed, integrated, and scaled using MOSFLM [ 32 ].

The crystal structure of σ W 4 /-35 W was determined by the molecular replacement (MR) method using PHASER [ 33 ]. The structure of the E . coli σ E 4 /-35 element (PDB ID: 2H27) was used as a template for MR. MR solution was found from the truncated σ E 4 (residues 127–186)/-35 element. Cycles of refinement and model building were performed at 3.1 Å resolution using PHENIX.refine [ 34 ] and COOT [ 35 ]. Final refinement resulted in R / R free values of 24.8 / 29.0% without residues in the disallowed region of the Ramachandran plot. The data collection and refinement statistics are summarized in Table 1 . The final coordinates and structure factors were deposited in the Protein Data Bank (PDB ID: 6JHE). Structural alignment was performed using the DALI server [ 36 ]. Protein-ligand interactions were analyzed with LigPlot+ [ 37 ] and PDBePISA [ 38 ]. The free energy change (Δ G ) of the protein caused by ligand binding was analyzed using PDBePISA [ 38 ]. The conversion of Δ G to a dissociation constant ( K d) was calculated using the equation K d = e (Δ G /RT ) (R = 1.987 cal/molK; T = 293K). DNA geometry was analyzed using w3DNA [ 39 ]. Surface charge distribution was calculated using APBS [ 40 ]. The figures were drawn using PyMOL [ 41 ] and ALSCRIPT [ 42 ].

Accession number

The final coordinates and structure factors were deposited in the Protein Data Bank (PDB ID: 6JHE for σ W 4 /-35 W ).

Results and discussion

Overall structure

B . subtilis σ W 4 (residues 125–187) recognizes a cognate -35 promoter element ( Fig 1A and 1B ). Its consensus sequence is identified as T -36 G -35 A -34 A -33 A -32 C -31 X -30 T -29 T -28 T -27 , based on the promoter sequences of the SigW regulon [ 43 ]. σ W 4 was purified under high salt conditions (1 M NaCl) to minimize its instability, and dialyzed in low salt buffer together with the double-stranded DNA of A -38 T -37 T -36 G -35 A -34 A -33 A -32 C -31 C -30 T -29 T -28 T -27 (-35 W ) to allow σ W 4 binding to -35 W . Crystals of σ W 4 /-35 W belonging to a hexagonal space group grew under conditions containing PEG1500 and isopropanol as protein precipitants. Diffraction data were collected at a resolution of 3.1 Å ( Table 1 ), and the structure was determined by molecular replacement using the structure of a truncated E . coli σ E 4 /-35 E (-35 promoter element for SigE binding) as a template [ 8 ].

The crystal structure contains a σ W 4 monomer and a double-stranded -35 W in the asymmetric unit. Residues 134–186 of SigW and 11 nucleotide pairs of -35 W were traced into the electron density ( S1 Fig ) and the final structure was refined at R / R free values of 24.8 / 29.0% ( Table 1 ). σ W 4 is comprised of four α-helices (α6–α9) in the crystal structure of the SigW/RsiW complex [ 23 ]. However, residues 125–133, which correspond to α6, are disordered in the crystal structure of σ W 4 /-35 W ( Fig 1A ). The residues on α6 are likely to be flexible because they are not bound to DNA directly. α8–α9 of σ W 4 forms the HTH motif, and α9 is inserted into the major groove of -35 W as a DNA recognition helix ( Fig 1C and 1D ) [ 7 ]. Positively-charged residues are distributed on the DNA binding surface of α8–α9, whereas hydrophobic patches are distributed on the opposite side that interacts with σ W 2 in the crystal structure of SigW/RsiW ( Fig 1E and 1F ) [ 23 ].

Interactions between σ W 4 and the -35 promoter element

The recognition helix α9 mediates the major interactions between σ W 4 and -35 W through hydrogen bonds and hydrophobic interactions. The bases and backbones of three purine nucleotides (G -35 and A -34 in the non-template strand and G’ -31’ in the template strand; ‘ indicates the template strand of DNA) form hydrogen bonds with the residues on the N-terminal half of α9 in σ W 4 ( Fig 2A–2C and S2 Fig ). The guanine oxygen (O6) and backbone phosphate (OP2) of G -35 form hydrogen bonds with side chain amino groups (NH2) of R175 and R172, respectively. The purine nitrogen (N7) and backbone phosphate (OP2) of A -34 interact with the side chain oxygens (OG1) of T171 and T168. The guanine oxygen (O6) of G’ -31 forms a hydrogen bond with the side chain amino group (NZ) of K170. The electron density map for the side chain of K170 is relatively weak; however, the most-preferred rotamer is at hydrogen bond distance to the O6 of G’ -31 ( S2B Fig ). Hydrophobic interactions are observed between K170-G’ -31 T’ -32 , T171-A -34 , H174-T’ -32 T’ -33 , and R175-T -36 ( S3 Fig ).

In addition to α9, α7-α8 contributes to -35 W binding without base specificity. The backbone atoms (OP1, OP2, and OP2) of G’ -30 form hydrogen bonds with the side chain amino group (NZ) of K148, the side chain oxygen (OG) of S154, and the backbone nitrogen of L155, respectively ( Fig 2B and 2C ). Altogether, α9 in σ W 4 specifically recognizes -35 W , and α7-α8 provides additional contacts, leading to a tighter interaction.

Structural comparison of σ W 4 /-35 W and σ E 4 /-35 E

SigE is an E . coli ECF sigma factor activated in response to envelope stress and induces transcription of heat shock proteins [ 45 ]. Its σ 4 domain (σ E 4 ) recognizes the -35 element, of which the consensus sequence is G -35 G -34 A -33 A -32 C -31 T -30 T -29 (-35 E ) [ 46 ]. The σ E 4 structure is highly similar to σ W 4 [ 8 ]. σ W 4 is superimposed on σ E 4 with a root mean square deviation (RMSD) value of 1.8 Å for 53 Cα atoms ( S4A Fig ). The overall fold is conserved between σ W 4 and σ E 4 and the main differences are observed at the N- and C-termini ( S4A and S4B Fig ).

Overall, the interactions of σ E 4 and σ W 4 with the corresponding -35 elements are conserved. Three nucleotides, G -35 , G -34 , and G’ -31 , in -35 E , which correspond to G -35 , A -34 , and G’ -31 in -35 W , mediate purine nucleotide-specific interactions with the recognition α-helix ( Fig 2A ). The backbone and base of G -35 (G -35 in σ W 4 ) form hydrogen bonds with R173 (R172 in σ W 4 ) and R176 (R175 in σ W 4 ). The backbone and base of G -34 (A -34 in -35 W ) form hydrogen bonds with T169 (T168 in σ W 4 ) and S172 (T171 in σ W 4 ). Although the -34 position is not identical between -35 E and -35 W (G -34 in -35 E and A -34 in -35 W ), hydrogen bonds mediated by the backbone phosphate and purine N7 are conserved. The base of G’ -31 forms a hydrogen bond with R171 (R170 in σ W 4 ). The backbone phosphate of G’ -31 also forms a hydrogen bond with R178, which is not observed in the σ W 4 /-35 W structure. A’ -30 in-35 E mediates backbone interactions similarly to G’ -30 in -35 W ( Fig 2 ). The backbone phosphate of A’ -30 forms hydrogen bonds with the NH1 of R149 (K148 in σ W 4 ) and the backbone nitrogen of Y156 (L155 in σ W 4 ) ( Fig 2D and 2E ). Interaction between S154 and A’ -30 in σ W 4 /-35 W is missing in the σ E 4 /-35 E structure. In summary, purine base-specific hydrogen bonds in the structures of σ W 4 /-35 W and σ E 4 /-35 E are conserved, whereas the hydrogen bonds with the nucleotide backbone are slightly different.

Hydrophobic interactions between σ E 4 and -35 E are mostly conserved in the σ W 4 /-35 W structure ( S3 Fig ). Residues R171, F175, and R176 in σ E 4 (K170, H174, and R175 in σ W 4 ) interact with T’ -32 , T’ -33 , and C -36 in -35 E (T’ -32 , T’ -33 , and T -36 in -35 W ), respectively. Hydrophobic interactions between P166/G168/T169 and G -34 and between Y156 and A’ -30 are observed only in σ E 4 /-35 E , whereas the interaction between K170 and G’ -31 in σ W 4 /-35 W is observed only in σ E 4 /-35 E . A cation-π interaction is observed between R176 and the pyrimidine ring of C’ -34 in the structure of σ E 4 /-35 E . However, the distance between the base (T -36 ) and the corresponding residue (R175) is too far to form a cation-π interaction in the structure of σ W 4 /-35 W ( S5 Fig ).

DNA geometry of -35 W

Nucleotides A -33 A -32 in -35 E (G -35 G -34 A -33 A -32 C -31 T -30 T -29 ) do not form hydrogen bonds with σ E 4 , although these nucleotides are conserved among the -35 elements of the SigE regulon, and mutating these nucleotides in the Salmonella enterica serovar Typhimurium SigE has been shown to lead to defective transcription [ 47 ]. These nucleotides are involved in characteristic oligo(dA)/oligo(dT)-like DNA geometry that is rigid and straight with a narrow minor groove [ 8 , 48 ]. A previous structural study of the σ E 4 /-35 E complex suggested that the geometry of the narrowed minor groove is critical for σ E 4 recognition [ 8 ], and we show that -35 W also displays a narrowed minor groove ( Fig 3 ). Like -35 E , the narrowing of the minor groove of -35 W begins at A -33 A -32 and is stabilized downstream of the -35 element, even though the downstream sequence of -35 W has an insertion of two cytosines (A -33 A -32 C -31 C -30 T -29 ) ( Fig 3 ). In contrast, -G -33 A -32 in -35 A (the -35 element of Thermus aquaticus SigA) has a wider minor groove than normal B-DNA ( Fig 3 ). The crystal structure of σ W 4 /-35 W supports the suggestion that the A -33 A -32 conservation in the -35 element for group IV sigma factors is critical for the formation of the narrow minor groove [ 8 ].

Structural comparison of σ W 4 /-35 W and SigW/RsiW

The crystal structure of SigW complexed with anti-sigma RsiW was previously reported [ 23 ]. The structures of σ W 4 under the binding of -35 W and RsiW superimpose with an RMSD value of 1.6 Å for 53 Cα atoms ( S4B Fig ). Although conformational differences of σ W 4 in the structures bound to -35W and RsiW are minor, σ W 4 bound to -35 W exists in a slightly compact conformation. σ W 4 interacts with -35 W through the residues K148, S154, L155, T168, K170, T171, R172, and R175, with a surface area of 621.2 Å 2 buried at the binding interface ( Fig 4A and 4B ). σ W 4 interacts with RsiW through the residues I150, K170, H174, R177, E178, R181, R185, and L187, these interactions result in an approximately 50% larger burial of surface area (915.2 Å 2 ) ( Fig 4C and 4D ). The surface area of σ W 4 that binds -35 W and RsiW partially overlaps with residue K170 on σ W 4 ( Fig 4A and 4C ), indicating that the interactions of -35 W and RsiW with σ W 4 are mutually exclusive.

In the crystal structure of the SigW/RsiW complex, the -10 element-binding surface of σ W 2 is buried in the surface of σ W 4 [ 23 ], whereas the -35 element-binding surface of σ W 4 is directly blocked by RsiW ( Fig 4 ), suggesting that SigW inhibition by RsiW competes with -35 element binding in the promoter. The binding of the -35 element to σ W 4 results in a Δ G of -10.5 kcal/mol, which corresponds to a K d value of 1.47 x 10 −8 M. RsiW binding to SigW results in a surface burial of 1619.6 Å 2 , and a Δ G of -17.85 kcal/mol. The free energy change corresponds to a K d value of 4.84 x 10 −14 M. These observations indicate that DNA binding of SigW is structurally repressed in the presence of RsiW.

Role of conserved residues in σ W 4

B . subtilis contains multiple ECF sigma factors that respond to diverse environmental stresses. The positions L137, L147, and E157 of σ W 4 are highly conserved in B . subtilis ECF sigma factors ( Fig 5A and 5B ). The highly conserved residues are likely to be involved in the intrinsic folding of SigW, but not in DNA binding. L137 and L147 stabilize σ W 4 as part of the central innermost hydrophobic cluster ( S6A and S6B Fig ). E157 is associated with σ W 2 binding in the crystal structure of the SigW/RsiW complex ( S6C and S6D Fig ).

The residues involved in DNA binding are less conserved than those involved in intrinsic folding. Residues K170, T171, and R175, which mediate purine-specific hydrogen bonds in σ W 4 , are aligned to K/R, S/T, and K/R in B . subtilis ECF sigma factors, as well as E . coli SigE ( Fig 5A and 5B ). The conservation of these residues correlates with the three nucleotides that mediate purine-specific interactions (G -35 , A -34 , and G’ -31 in -35 W ). For example, A -34 in the SigV promoter interacts with T144 and T147, as does A -34 in -35 W , and T168/T171 in σ W 4 , whereas C -34 in the SigZ promoter is aligned with G138 and S141. These observations suggest that sequence variation in the DNA-binding interface confers the specificity required to discriminate between -35 elements ( Fig 5C ). In summary, conserved residues in B . subtilis ECF σ 4 are mainly involved in intramolecular stability and interactions with the -35 element. Slight sequence variations in the residues involved in the binding of the -35 element may contribute to the fine-tuning of the promoter selectivity and binding affinity for individual ECF sigma factors.

Conclusions

Group IV ECF sigma factor is activated in response to environmental stress and initiates transcription of its own regulon to counter that stress. The crystal structure of σ W 4 /-35 W shows that SigW selectively recognizes cognate -35 promoter elements which have a narrowed minor groove, in a similar manner to the E . coli SigE. Comparison with the SigW/RsiW structure shows that SigW binding to the -35 promoter element and anti-sigma RsiW is mutually exclusive. These results provide the structural basis for the mechanism of SigW activation, and improve our understanding of the selective interactions between σ 4 domains and their cognate -35 promoter elements.

Supporting information

Overall electron density map of σ W 4 /-35 W .

Red and orange stick models indicate σ W 4 and -35 W , respectively. 2Fo-Fc maps are drawn at two different orientations. The contour level is set to 1.0 σ.

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Map of electron density around the binding interface in the σ W 4 /-35 W structure.

2Fo-Fc maps are drawn at same scale and orientation as those for models in Fig 2C and arranged in the same order as the panels in Fig 2C . The contour level is set to 1.0 σ.

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Hydrophobic interactions between the σ 4 domain and the -35 promoter element.

(A, B) Hydrophobic interactions between σ W 4 and -35 W shown at two different orientations. Residues and nucleotides, which are associated with hydrophobic bonds, are drawn as red and orange stick models. Dotted lines indicate hydrophobic interactions. (C, D) 2Fo-Fc electron density maps in (C) and (D) are shown at same scale and orientation as those for models in S2A and S2B Fig , respectively. The contour level is set to 1.0 σ. (E, F) Hydrophobic interactions between σ E 4 and -35 E . Residues and nucleotides which are associated with hydrophobic bonds are drawn as purple and green stick models.

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Structural comparison of σ 4 domains.

(A) Superposition of σ E 4 /-35 E and σ W 4 /-35 W structures. N- and C-termini of σ W 4 are labeled. (B) Distribution of root-mean-square values between Cα positions of superimposed σ E 4 and σ W 4 structures. (C) Superposition of σ W 4 domains from the structures of σ W 4 /-35 W and SigW/RsiW.

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Cation-π interaction.

(A) The green dotted line indicates the cation-π interaction between R176 of σ E 4 (cyan model) and C -36 of 35 E (green). The corresponding residue in σ W 4 (R175) and base in -35 W (T -36 ) are drawn as a stick model. The distance between the Arg and pyrimidine is labeled.

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Conserved residues of σ 4 domains.

The residues that participate in intramolecular interactions with L137 (A), L147 (B), or E157 (C) are drawn as stick models and the residue number is labeled. The helix and loop in σ W 4 are colored pink and light brown. (D) σ W 4 in SigW/RsiW structure (PDB ID: 5WUQ) is superposed onto that of the σ W 4 /-35 W structure. SigW in SigW/RsiW structure is colored green. E157 in σ W 4 interacts with T71 in σ W 2 and does not participate in DNA binding.

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Data Availability

The final coordinates and structure factors were deposited in Protein Data Bank (PDB ID: 6JHE).

Funding Statement

DYK was supported by the Yeungnam University Research Grant. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Associated Data

Supplementary Materials

Overall electron density map of σ W 4 /-35 W .

Red and orange stick models indicate σ W 4 and -35 W , respectively. 2Fo-Fc maps are drawn at two different orientations. The contour level is set to 1.0 σ.

(TIF)

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Map of electron density around the binding interface in the σ W 4 /-35 W structure.

2Fo-Fc maps are drawn at same scale and orientation as those for models in Fig 2C and arranged in the same order as the panels in Fig 2C . The contour level is set to 1.0 σ.

(TIF)

Click here for additional data file.

Hydrophobic interactions between the σ 4 domain and the -35 promoter element.

(A, B) Hydrophobic interactions between σ W 4 and -35 W shown at two different orientations. Residues and nucleotides, which are associated with hydrophobic bonds, are drawn as red and orange stick models. Dotted lines indicate hydrophobic interactions. (C, D) 2Fo-Fc electron density maps in (C) and (D) are shown at same scale and orientation as those for models in S2A and S2B Fig , respectively. The contour level is set to 1.0 σ. (E, F) Hydrophobic interactions between σ E 4 and -35 E . Residues and nucleotides which are associated with hydrophobic bonds are drawn as purple and green stick models.

(TIF)

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Structural comparison of σ 4 domains.

(A) Superposition of σ E 4 /-35 E and σ W 4 /-35 W structures. N- and C-termini of σ W 4 are labeled. (B) Distribution of root-mean-square values between Cα positions of superimposed σ E 4 and σ W 4 structures. (C) Superposition of σ W 4 domains from the structures of σ W 4 /-35 W and SigW/RsiW.

(TIF)

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Cation-π interaction.

(A) The green dotted line indicates the cation-π interaction between R176 of σ E 4 (cyan model) and C -36 of 35 E (green). The corresponding residue in σ W 4 (R175) and base in -35 W (T -36 ) are drawn as a stick model. The distance between the Arg and pyrimidine is labeled.

(TIF)

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Conserved residues of σ 4 domains.

The residues that participate in intramolecular interactions with L137 (A), L147 (B), or E157 (C) are drawn as stick models and the residue number is labeled. The helix and loop in σ W 4 are colored pink and light brown. (D) σ W 4 in SigW/RsiW structure (PDB ID: 5WUQ) is superposed onto that of the σ W 4 /-35 W structure. SigW in SigW/RsiW structure is colored green. E157 in σ W 4 interacts with T71 in σ W 2 and does not participate in DNA binding.

(TIF)

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Data Availability Statement

The final coordinates and structure factors were deposited in Protein Data Bank (PDB ID: 6JHE).