Key Features of Σs Required for Specific Recognition by Crl, a Transcription

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Key features of σS required for speciﬁc recognition by Crl, a transcription factor promoting assembly of RNA polymerase holoenzyme

Amy B. Bantaa, Robert S. Chumanovb, Andy H. Yuanc, Hueylie Lina, Elizabeth A. Campbelld, Richard R. Burgessb, and Richard L. Goursea,1

aDepartment of Bacteriology and bMcArdle Laboratory for Cancer Research, University of Wisconsin–Madison, Madison, WI 53706; cDepartment of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115; and dLaboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065

Edited by Jeffrey W. Roberts, Cornell University, Ithaca, NY, and approved August 15, 2013 (received for review June 18, 2013)

Bacteria use multiple sigma factors to coordinate gene expression connected by flexible linkers (3). The most highly conserved do- in response to environmental perturbations. In Escherichia coli and main, σ2 (composed of homology regions 1.2–2.4), interacts with other γ-proteobacteria, the transcription factor Crl stimulates σS- the β′ clamp helices (β′CH) in core RNAP (residues 262–309, also dependent transcription during times of cellular stress by promot- known as the β′ coiled coil) (15), and σ4 interacts with the flap ing the association of σS with core RNA polymerase. The molecular domain of β (16). Free sigma factors undergo conformational basis for specific recognition of σS by Crl, rather than the homol- changes to associate with core RNAP and then undergo further ogous and more abundant primary sigma factor σ70, is unknown. conformational changes in the context of holoenzyme to bind to the Here we use bacterial two-hybrid analysis in vivo and p-benzoyl- promoter and to melt the DNA in the vicinity of the −10 element phenylalanine cross-linking in vitro to define the features in σS and transcription start site (15, 17–20). S responsible for specific recognition by Crl. We identify residues Direct interaction of Crl and σ in E. coli and Salmonella S S enterica fi in σ conserved domain 2 (σ 2) that are necessary and sufficient was reported previously in vitro by gel ltration (14), to allow recognition of σ70 conserved domain 2 by Crl, one near surface plasmon resonance (SPR) (12), and bacterial two-hybrid the promoter-melting region and the other at the position where (BTH) analysis (12, 21). BTH analysis further suggested that a large nonconserved region interrupts the sequence of σ70.We S. enterica Crl interacts with fragments containing conserved σS σS then use luminescence resonance energy transfer to demonstrate domain 2 of ( 2) (21). Although most activators interact with directly that Crl promotes holoenzyme assembly using these spec- sigma by recognizing domain 4 (reviewed in refs. 2, 22), two ificity determinants on σS. Our results explain how Crl distin- activators exhibiting no sequence similarity to Crl [Chlamydia guishes between sigma factors that are largely homologous and trachomatis GrgA (23) and Streptomyces coelicolor RbpA (24)] activates discrete sets of promoters even though it does not bind were recently shown to interact with domain 2 of the primary to promoter DNA. sigma factor, suggesting that such interactions may be more widespread than appreciated previously. E. coli σS RNAP formation | transcription initiation | bacterial stress response | Here we investigate the interaction between and Crl fi RpoS | curli fiber to gain insight into its mechanism(s) of action. We de ne two S surface-exposed patches on σ 2 needed for specific recognition 70 S by Crl; create a σ 2–σ 2 chimera to show that these residues are ranscription initiation in bacteria requires the assembly of fi σS σ both necessary and suf cient for Crl to distinguish from other Ta sigma factor ( ) with the RNA polymerase (RNAP) cata- σ70 α β β′ sigma factors, including ; and demonstrate directly that Crl lytic core (E, composed of 2 -subunits and one each of , , and functions as a holoenzyme assembly factor by using these spec- ω σ BIOCHEMISTRY ) to form RNAP holoenzyme (E ), which in turn recognizes ificity determinants on σS. We thereby determine how this promoter sequences (1) (reviewed in ref. 2). Multiple sigma factors compete for binding to core RNAP (reviewed in refs. 3, Significance 4), and each sigma factor controls a specific set of promoters. In Escherichia coli, which has seven sigma factors, σ70 is the primary sigma, and σS is important for certain stress responses Bacteria can change the relative amounts of different sigma and during the stationary phase of growth (5). EσS-dependent factors that associate with the RNA polymerase (RNAP) cata- transcription initiation is regulated by σS, whose concentration is lytic core enzyme, thereby altering the relative amounts of the itself regulated at the levels of transcription, translation, and RNAP holoenzymes and modifying gene expression globally protein stability (reviewed in ref. 6). EσS-dependent transcription under changing nutritional or environmental conditions. We is also activated by Crl (7), a small protein that increases expres- show here that the Escherichia coli transcription factor Crl sion of many stress response genes and those required for forma- increases assembly of the holoenzyme containing the stress tion of amyloid curli fibers (which accounts for its name) involved response sigma factor, sigma S. We identify the key determi- in adhesion and biofilm formation (reviewed in refs. 2, 6, 8). nants necessary for Crl to distinguish between sigma S and S The effect of Crl on σ -dependent transcription in vivo is most other sigma factors, thereby specifically promoting the sigma S pronounced during the transition into stationary phase (9). It has transcriptional program. been proposed that Crl functions by increasing the concentration S S of Eσ holoenzyme by facilitating assembly of σ with core Author contributions: A.B.B., R.S.C., A.H.Y., R.R.B., and R.L.G. designed research; A.B.B., RNAP (10–12) because Crl’s effects on transcription are greatest R.S.C., A.H.Y., H.L., and E.A.C. performed research; A.B.B. contributed new reagents/ in vitro when the concentration of σS is lowest, and over- analytic tools; A.B.B., R.S.C., A.H.Y., H.L., E.A.C., R.R.B., and R.L.G. analyzed data; and expression of σS complements a crl deletion in vivo (13). Effects A.B.B. and R.L.G. wrote the paper. of Crl have also been reported on postholoenzyme assembly The authors declare no conflict of interest. steps including promoter binding (14) and open complex for- This article is a PNAS Direct Submission. mation (12). 1To whom correspondence should be addressed. E-mail: [email protected]. Sigma factors contain several protease-resistant domains, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. each composed of subregions defined by sequence conservation, 1073/pnas.1311642110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1311642110 PNAS | October 1, 2013 | vol. 110 | no. 40 | 15955–15960 Downloaded by guest on September 24, 2021 S nonconventional transcriptional activator distinguishes between these variants (representing 30 different positions in σ 2) were S S sigma factors to stimulate σ -dependent gene expression. unique (Table S1). Second, we tested the 39 σ 2 variants for interaction with the β′CH fragment to identify those that were Results defective in binding to Crl but that still bound to core RNAP. E. coli σS S Crl Interacts with Conserved Domain 2 in . It was reported The interaction between σ 2 and Crl was reduced (<65% of that S. enterica σS fi S recently that Crl interacts with 2 (21). To con rm with wild-type σ 2) in 15 of the variants that still interacted with E. coli E. coli S that this interaction is conserved in ( Crl has 83% core RNAP (>51% of that with wild-type σ 2)(Fig. S2A and identity/92% similarity to S. enterica Crl, and E. coli σS has 99% Table S1). Substitutions at four positions that were not con- identity/99% similarity to S. enterica σS), we constructed a library served between σS and σ70, D87, D135, P136, and E137 (teal in S S of σ fragments based on the predicted endpoints of its stable Fig. 2A and Fig. S2A, shaded rows in Table S1), disrupted the σ 2 domains and tested their interactions with Crl using a BTH assay interaction with Crl (<40% of the wild type) without dramati- S σS β′ (25). Crl interacted with the σ 2 fragment and with larger frag- cally perturbing the 2 interaction with the CH (gray in Fig. S A fi ments encompassing σ 2, and it did not interact with fragments 2 ), suggesting that these residues represent speci c determi- S lacking σ 2 (Fig. 1). As the lack of detectable protein–protein nants for Crl binding. We also note that many other substitutions interactions in the BTH assay could be attributable to misfolding near D87 were obtained in the screen for amino acid residues S A or instability of σ 2 fusion proteins, we verified that all fragments altering Crl binding (Fig. S2 and Table S1), but these sub- of σS used in our study interacted with fragments of core RNAP stitutions also diminished the interaction with the β′CH, com- containing either the β′CH or the β-flap (Fig. S1A). plicating the interpretation of the role of these residues in We also investigated the interaction of Crl with other sigma Crl binding. S factors. Previous in vitro transcription data suggested that Crl may We constructed a structural homology model of σ 2 based on have small effects on transcription by Eσ70 and EσH (10) but an the X-ray crystal structure of σ70 (1SIG) and the alignment in 70 B C–F σS interaction between Crl and σ was not detected by SPR (12), Fig. 2 (Fig. 2 and refs. 26, 27). 2 has an elongated, and genome-wide expression analysis of an E. coli crl deletion asymmetric shape, with D87, D135, P136, and E137 all surface strain showed few differences from wild-type (WT) in the ab- exposed and located on the same face at one end of the domain. S 70 sence of σS under the conditions tested (11). To address directly The D135–P136–E137 cluster in σ (E420–Y421–R422 in σ ), whether Crl interacts with other σ70 family σ-factors (σ70, σH, σF, called the “DPE motif” hereafter, is surrounded by residues that E FecI σ , and σ ), we examined fragments corresponding to σ2 from are highly conserved between the two sigma factors. Although each of the σ70 family σ-factors for interaction with Crl in vivo D87 is not contiguous with the other three residues, it is sepa- in the BTH assay, again using their interaction with the β′CH as rated from the DPE motif by only ∼17–18 Å (Fig. 2 E and F). A σS B C Crl-sized globular protein should be able to span the DPE motif a positive control. Crl interacted only with 2 (Fig. S1 and ). S These results suggest that Crl distinguishes between σ-factors and D87 on σ . σS – through its differential interactions with conserved domain 2 of D87 is within a stretch of residues (A83 A89) that is not σ70 σS, the promoter DNA binding and melting domain. conserved in but corresponds to the position of the nonconserved region (NCR) in σ70, a 252-aa section (I123–R374) S that links homology regions 1.2 and 2.1. The NCR is not present Identification of Residues on the Surface of σ 2 Recognized by Crl. To S in alternative sigma factors, and its sequence differs even among determine residues in σ 2 necessary for interaction with Crl, we used a two-step BTH screen. First, we generated a random li- primary sigma factors in diverse bacterial species. We suggest S below that the binding site for Crl on the surface of σS includes brary of σ 2 fragments and screened the resulting collection of σS variants for altered interaction with Crl. About 10% of the both the DPE motif and the region corresponding to where the 2 NCR is positioned in σ70. colonies exhibited lighter blue or white colony color on X-gal S S We screened a library of σ 2 fragments with other substitutions plates. About 50% (74) of the 144 σ 2 variant-encoding plasmids that were sequenced contained single substitutions, and 39 of at positions D87, D135, or E137 for interactions with Crl and with the β′CH using the BTH assay (Table S2 and Fig. S2B). Only fragments with the wild-type residues at all three positions resulted in wild-type interactions with Crl. D87 or D135 sub- fusion protein stitutions had larger effects on Crl interactions than on β′CH s AA interactions. Of the substitutions at E137, only E137D retained S domain residues -Crl Interaction a substantial interaction with Crl (40% of the wild-type level), whereas the interaction with the β′CH tolerated at least 11 dif- 1-4 (1-330) ferent substitutions, strongly suggesting that the charge of E137 is crucial for Crl recognition. 1-3 (1-221) S 1-2 (1-163) Crl Function Requires E137 in the Context of Full-Length σ . We tested the importance of the DPE motif for activation of tran- 2 (57-163) scription by Crl in vivo in full-length σS by measuring transcription from an osmY promoter–lacZ fusion in strains with WT σS, 2-3 (57-221) ΔrpoS cells (null), and cells with the chromosomally encoded 2-4 (57-330) rpoS substitutions D135Q, E137A, or E137Q (Fig. 3A). osmY CI fusion protein promoter activity depended on rpoS, and it was stimulated ap- 3-4 (164-330) A Crl proximately threefold by Crl (Fig. 3 , null versus WT), consistent with results from previous studies (28). In the absence of 4 (243-330) control Crl, the activity of the osmY promoter was slightly lower in the (unfused control) rpoS mutant strains than in the WT strain, but the promoter activity was still much higher than in the rpoS null strain (Fig. 3A, 0 250 500 750 1000 1250 open bars). However, Crl had no effect on osmY promoter ac- rpoS A fi -galactosidase activity (Miller Units) tivity in the mutant strains (Fig. 3 ,compare lled and open bars). We conclude that the holoenzymes containing σS(E135Q), S(E137A) S(E137Q) S S σ σ Fig. 1. Crl interacts with the DNA-melting domain of σ (σ 2). BTH analysis ,and are transcriptionally competent, but they of the interaction of σS fragments with Crl (black) or the unfused control do not respond to Crl in vivo. (light gray) by measurement of β-galactosidase activity. Error bars show SD We also examined transcription activation by Crl in vitro to S between two separate experiments of two independent cultures each. determine whether the effect of the site on σ predicted to

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CDE F

S S Fig. 2. Identification of σ 2 residues needed for interaction with Crl (BTH analysis). (A) σ 2 variants with reduced interaction with Crl (<0.40 relative to WT; black bar, WT and teal bars, variants) that retain an interaction with the β′CH (>0.51 relative to WT; gray bars) and whose identities are different in σS and σ70. Error bars: SD between ratio of variant/WT β-galactosidase activity in two separate experiments of two independent cultures each. Complete results of BTH S 70 screen are shown in Table S1 and Fig. S2A.(B) Amino acid sequence alignment of σ 2 and σ 2. Identity (j), similarity (:); residues identified in A are colored teal. Colored bar indicates σ70 regions defined by homology [region 1.2 (blue), nonconserved region (green), 2.1 (yellow), 2.2 (orange), 2.3 (red), and 2.4 (purple)]. S 70 S (C and D) Homology model of σ 2 based on σ structure (1SIG) with colors as in B.(E and F) Surface representation of σ 2 from C and D; residues with Crl interaction <0.4 relative to WT are in teal, as in A.

bind Crl is direct. Crl was incubated with full-length σS(WT) or E137) and in the BPA-mediated cross-linking assay (Y78, F79, σS(E137Q) before addition of core RNAP and NTPs. Crl increased and R93) (Fig. 4B). transcription from the osmY promoter by EσS(WT) RNAP ∼2.3-fold 70 S at the highest concentration of Crl tested but did not affect A σ 2 Chimera Containing the Key Determinants from σ 2 Binds Crl. S(E137Q) S transcription by Eσ RNAP (Fig. 3 B and C). The basal The patch on the surface of σ 2 necessary for interaction with Crl activity of σS(E137Q) is lower than wild-type, which could result is composed of residues from two noncontiguous regions where S 70 S from differences in the active fractions of the puriﬁed proteins or σ 2 and σ 2 differ: σ D87, which is in the stretch of residues from direct effects of E137Q on transcription initiation. Taken surrounding the corresponding position in σ70 where the NCR is together, these data indicate that Crl does not activate tran- found, and the σS DPE motif adjacent to the promoter-binding scription by RNAP containing σS(E137Q) and that the effect of region (Fig. 5A). 70 S this substitution on function is direct. We created a triple substitution in σ 2 corresponding to the σ 70 70 DPE motif [σ 2 (DPE), i.e., σ E420D/Y421P/R422E] (Fig.

S 70 BIOCHEMISTRY Crl Cross-Links Directly to the Genetically Identified Surface on σ . To 5B). σ 2 (DPE) interacted with the β′CH but not with Crl in the determine whether the genetically identified activation patch on BTH assay (Fig. 5C), indicating that the DPE motif is not suf- S S the surface of σ 2 interacts directly with Crl, we incorporated the ficient for Crl recognition. In contrast, the σ DPE motif in photoreactive cross-linker p-benzoyl-phenylalanine (BPA) into combination with a 263-aa deletion of the σ70 NCR and flanking specific sites in σS using a nonsense suppressor tRNA/tRNA residues (G115–E378) and its replacement with 19 amino acids fi σS S 70 synthetase system (29). Puri ed variants with single BPA (E75–R93) from the corresponding region in σ [σ 2 (DPE) substitutions at positions in the vicinity of, but not overlapping, ΔNCR19] (Fig. 5B) interacted strongly with both the β′CH and D87 or the DPE motif (A73, E74, Y78, F79, R81, R82, L84, R91, with Crl (Fig. 5C), indicating that the DPE motif together with R93, R138, R141, and R145) were chosen for analysis so as to the 19 amino acids flanking the location of the NCR is sufficient S avoid disruption of the predicted Crl–σ 2 interface. An addi- for Crl recognition. tional variant with a BPA substitution (R108) located in a part of To further refine our understanding of the specificity deter- S S 70 S σ 2 far from the predicted interface was constructed as a control. minants on σ , we also constructed σ 2–σ 2 chimeras with a 252- Radiolabeled Crl was incubated with the BPA-containing σS var- aa NCR deletion (I123–R374) replaced with 7 amino acids S S 70 iants, exposed to UV light, and the cross-linked σ –Crl complexes (E83–R87) from σ with or without the DPE substitution (σ 2 70 were analyzed by SDS/PAGE and phosphorimaging. BPA sub- DPE ΔNCR7 and σ 2 ΔNCR7, respectively) or just the 19-aa S 70 70 stitutions at either of two positions in σ , Y78 or F79, cross-linked substitution alone (σ 2 ΔNCR19). The σ 2 DPE ΔNCR7 vari- efficiently to 32P–Crl, a BPA at position R93 cross-linked less ef- ant interacted with the β′CH but not with Crl in the BTH assay ficiently, and BPA substitutions at the other 10 positions did not (Fig. S4B), suggesting that the D87 patch requires additional cross-link to Crl (Fig. 4A). To confirm the identities of proteins in residues outside the 7-aa region but within the 19-aa region in σS the cross-linked complexes, reciprocal experiments were also per- to contact Crl or to position D87 to contact Crl. The ΔNCR formed with unlabeled Crl and radiolabeled σS with BPA at Y78, chimeras without the σS DPE motif did not interact with the β′ F79, or R93. These studies confirmed that the gel bands identified CH (or Crl), suggesting that these fragments might be unstable. in the experiments with 32P–Crl corresponded to σS–Crl com- Taken together, the requirement for the σS DPE motif and for plexes (Fig. S3 B–E). We conclude that Crl and σS interact di- D87 for Crl binding (Fig. 2A and Fig. S2B), the formation of Crl S S rectly and that their interface includes or is adjacent to the σ 2 cross-links with Y78, F79, and R93 of σ (Fig. 4), and the re- 70 S residues identified in the BTH screen (D87, D135, P136, and quirement for the 19-aa NCR substitution in the σ 2–σ 2

Banta et al. PNAS | October 1, 2013 | vol. 110 | no. 40 | 15957 Downloaded by guest on September 24, 2021 σS A osmY promoter-lacZ Direct Demonstration That Crl Promotes Assembly of E Holoenzyme. 600 To address the mechanism by which Crl enhances transcription, we used a luminescence resonance energy transfer (LRET) assay 500 (30) to test whether Crl uses the surface described above for σS 400 promoting E holoezyme assembly. Core RNAP labeled with WT a donor fluorophore (terbium) and σS (wild type or E137Q vari- 300 Δcrl ant) labeled with an acceptor fluorophore (fluorescein) were in- σS 200 cubated together in the presence or absence of Crl. E holoenzyme assembly was determined after excitation, based on β -gal activity 100 the acceptor/donor ratio. The amount of EσS increased with the concentration of σS(WT) or σS(E137Q) (Fig. 6 A and B), but Crl only 0 enhanced assembly of EσS(WT). The largest effect of Crl (ap- proximately twofold) under these conditions was observed at 37 null WT rpoS allele σS C σ70 σH D135Q E137A E137Q nM (Fig. 6 ). Crl had no effect on E or E formation (Fig. 6D), consistent with the absence of Crl interactions with σ70 and H S B In vitro transcription σ (Fig. S1 B and C) and a model in which Crl functions as a σ - specific holoenzyme assembly factor. S (WT) S (E137Q) Discussion Crl (μM) Crl (μM) By helping σS bind to core RNAP and increasing the concen- 0 0.06 1.60.540.18 4.8 0 0.06 4.81.60.540.18 tration of EσS when σS concentrations are low, we propose that Crl favors σS in its competition with other sigma factors for core osmY RNAP leading to increased occupancy of σS-dependent promoters. Crl thereby speeds up the switch to the σS transcriptional fi C 3.0 program early in stress responses. Crl copuri es with RNAP under certain stress conditions (31), consistent with a model in which Crl 2.5 regulates transcription in response to specific nutritional or environmental signals. Although Crl has thus far been identified only 2.0 WT in the γ-proteobacteria, we suggest that there could be proteins 1.5 E137Q that perform analogous tasks in other organisms. There are at least two, nonexclusive explanations for how Crl 1.0 could promote RNAP assembly. First, free σ-factors must as- sume conformations that avoid proteolysis of the linker regions Transcription +/- Crl Transcription 0.5 that connect their stable domains, but they must also undergo 0 1 2 3 4 5 major conformational changes to associate with core RNAP [Crl] (µM ) (20). Crl could aid assembly by breaking intramolecular or σS–σS intermolecular interactions to facilitate interactions with core. Fig. 3. Crl activation of osmY promoter is dependent on the DPE patch in Second, Crl could interact with both σS and core RNAP and act vivo and in vitro. (A) osmY promoter–lacZ expression in early stationary as a bridge to facilitate formation of EσS. In support of this ∼ phase (ODA600 2.4). Error bars represent SD between two experiments of “molecular Velcro” model, a weak interaction was detected B two independent cultures each. ( ) Multiple round in vitro transcription previously between Crl and core RNAP, even in the absence of σS, osmY σS(WT) σS(E137Q) from plasmid-borne promoter (pRLG8941) by E or E in using SPR (12). the presence of a range of concentrations of Crl. Representative experiment The σ70 NCR has been implicated in promoter escape through is shown. (C) Effect of Crl on EσS(WT) (filled triangles) or EσS(E137Q) (open interactions with the β′ clamp (32). Because Crl binds near this circles) normalized to the same reaction without Crl. The plot shows the σS σS average and SD from three separate experiments (six reactions). position in , we speculate that Crl could aid E holoenzyme formation by facilitating interactions between analogous regions S in the β′ clamp and σ 2. However, Crl might also play roles at later steps in the transcription mechanism. In theory, a Crl interaction chimera (Fig. 5) indicate that the DPE motif and the surface of with β′ and σS could also play a role in EσS promoter escape. σS surrounding the position where the NCR is found in σ70, not Because our proposed Crl–σS interface is directly adjacent to the just removal of the NCR, are both required for Crl recognition area of σ forming critical interactions with promoter DNA of σS. (e.g., σS residue D135 is homologous to σ70 E420 and Thermus

BPA crosslinking S A B 2 homology model

s HMK-His- BPA crosslinks to Crl S -Crl BTH mutants E74 Y78 F79 R93 2 WT (BPA) (BPA) (BPA) (BPA) D87 UV (min) 0 10 0110010 0 0100 D135 Fig. 4. BPA-mediated cross-linking of σS to Crl. (A) Crl- s R93 P136 SDS/PAGE gel showing results of UV exposure on 32P–Crl incubated with WT and four different σS BPA F79 variants. Cross-linked 32P–Crl–σS complexes are in- S dicated. (B) σ 2 homology model showing positions of σS BPA variants that cross-link to Crl (orange) and 32 . E137 P Crl σS substitutions that disrupt Crl–σS interaction iden- Y78 tiﬁed in the BTH analysis (cyan).

15958 | www.pnas.org/cgi/doi/10.1073/pnas.1311642110 Banta et al. Downloaded by guest on September 24, 2021 A S and 70 comparison

Identical ( | ) promoter DNA- Similar ( : ) NCR Not conserved binding surface NCR/Region 1.2-2.1 linker

S homology model 70 2 structure (1SIG) ‘DPE’ 2

B 70(94-114) S(75-93) 70(379-419) S(135-137) 70(423-448)

‘ΔNCR19’ ‘DPE’ 70 S TTDPVRMYMREMGTVELLTREEEVYFARRALRGDVASRRRMVEANLRLVISIAKKYTNRGLQFLDLIQEGNIGLMKAVDKFDPERGYKFSTYATWWIRQAITRSIADQAR 2 - 2 chimera

C 1000 800 Interaction with: 600 Crl β.CH 400 control

200

β -galactosidase activity (MU) 0 o S 70 70 70 90 2 2 2 2 (DPE) (DPE) (ΔNCR19)

70 S S 70 S Fig. 5. σ variant with a σ DPE motif and an NCR deletion interacts with Crl. (A) Conservation between σ 2 and σ 2. Surface representation of σ 2 (homology model) 70 S 70 and σ 2 crystal structure (1SIG). Identities, light gray; similarities, dark gray; not conserved, purple; position of NCR, green. DPE motif (σ D135/P136/E137) and σ NCR 70 S S (I123–R374) are indicated. (B) Amino acid sequence of the σ 2–σ 2 chimera showing positions of the σ 2 substitution for the NCR (green) and ﬂanking residues (yellow) S S and the σ DPE motif (blue). The positions of these substitutions are illustrated on two views on the σ 2 homology model. (C) BTH analysis of interaction of σ2 variants with Crl (red) and β′CH (gray) compared with background level (white). Error bars show SD between two separate experiments of two independent cultures each.

aquaticus σA E243) (18, 19), another possibility is that Crl could σ70, respectively, to prevent assembly with core RNAP (33, 34), affect promoter binding or open complex formation, as sug- and DnaJ binds to and/or affects analogous surface(s) in σH to gested previously (12, 14), assuming that Crl stays associated influence its conformation, folding, and stability (35). with EσS in the transcription initiation complex. In summary, we have demonstrated directly that Crl functions We also note that the position where the NCR interrupts con- as a σS-specific RNAP holoenzyme assembly factor. A structure served domain 2 of σ70 and where Crl binds to σS might provide for a Crl-like protein from Proteus mirabilis has been reported BIOCHEMISTRY a unique recognition surface for other positive or negative reg- recently [see Protein Data Bank (PDB), http://www.rcsb.org/pdb/ ulators affecting transcription initiation. For example, the anti- explore.do?structureId=3RPJ]. In future studies, we will focus sigma factors RseA and Rsd bind to nearby surfaces on σE and on identification of the surface on Crl that interacts with σS,

ACS (WT) B S (E137Q) 37 nM S D

2.0 2.0 1.0 2.4 2.0 1.6 1.6 0.8 0.6 1.6 1.2 1.2 1.2 assem

assembly 0.4 - - - S - !""#$%&'( 0.8 0.2 E

assembly 0.8 assembly 0.8

S 0.4 S 0.0 E assmebly +/- Crl

E S 0.0 0.4 E 0.4 S WT E137Q S 70 H WT E137Q 0.0 0.0 0.1 1 10 100 1000 0.1 1 10 100 1000 S __ __ Crl (µM) 0 3 9 27 (nM) S (nM) + Crl - Crl

Fig. 6. Crl promotes assembly of EσS(WT) but not EσS(E137Q) in vitro. (A) Equilibrium binding of σS or (B) σS(E137Q) variant to core RNAP (10 nM) in the presence or absence of Crl (16 μM). EσS holoenzyme assembly represents the equilibrium LRET signal (acceptor/donor ratio) 1 h after mixing terbium-labeled core RNAP with ﬂuorescein-labeled σS.(C) Histogram showing core RNAP interaction with 37 nM σS ± Crl. Error bars indicate average of four samples. (D) Crl promotes assembly of EσS but not Eσ70 or EσH in vitro. Fold effect of Crl (0, 3, 9, and 27 μM) on RNAP holoenzyme assembly [interaction between ﬂuorescein-labeled σS, σ70,orσH (10 nM) and terbium-labeled core RNAP (8.3 nM)] using an LRET assay.

Banta et al. PNAS | October 1, 2013 | vol. 110 | no. 40 | 15959 Downloaded by guest on September 24, 2021 characterize the proposed Crl–EσS complex, and address the were expressed (42) and purified by Ni-affinity chromatography. Proteins mechanism(s) by which Crl promotes RNAP assembly and (po- were 32P-radiolabeled on an N-terminally encoded HMK recognition site using tentially) later steps in transcription initiation. HMK (15, 43). Photoactivated cross-linking of BPA-substituted proteins was performed in duplicate (42). Materials and Methods Additional details about methods and modifications from previously pub- BTH Analysis. Protein–protein interaction was determined as described pre- lished methods are provided in SI Materials and Methods. viously (25, 44).

Strains, Plasmids, and Oligonucleotides. A complete list of strains, plasmids, In Vivo Promoter Activity Assays. Strains harbored an osmY promoter–lacZ and oligonucleotides are provided in Tables S3–S5. Site-directed mutagenesis transcriptional fusion on a single copy λ-prophage, and strains with mutant of plasmids was performed using the Quikchange II kit (Stratagene). Ran- alleles, were created by P1 transduction. β-Galactosidase assays were per- dom mutagenesis was performed by PCR with Taq polymerase as described formed as for BTH assays. previously (33). Strains for BTH analysis were created by cotransformation of –lacZ a strain harboring a promoter reporter on an F episome with plasmids In Vitro Transcription Assays. Multiple round transcription using a plasmid –lacZ encoding bait and prey fusion proteins. Promoter transcriptional fusion template was performed as previously described (10). reporters are single copy λ-prophage constructed from a plasmid in- termediate as previously described (36). Gene deletions and allelic replace- In Vitro RNAP Assembly Assay. Random lysine labeling of proteins with ter- ments were made by λ-Red–mediated recombination (37) or were obtained bium chelate or fluorescein, purification and LRET assays were performed as from the Keio collection (38). Mutant alleles were transferred to the re- previously described (30). porter strains by transduction with P1vir (39). ACKNOWLEDGMENTS. We thank Mark Mandel and Tom Silhavy for advice E. coli σS Proteins. core RNAP was prepared, and untagged was prepared and materials and A. Hochschild, L. Westblade, J. Peters, P. Chandrangsu, – from inclusion bodies and refolded (40, 41). His Crl was overexpressed and T. Gaal, and W. Ross for many helpful comments. This work was supported purified using Ni-affinity and heparin chromatography. The tag was removed by National Institutes of Health (NIH) Grant R37 GM37048 (to R.L.G.), and in with thrombin. BPA-substituted heart muscle kinase (HMK)–His–σS variants part by NIH Predoctoral Training Grant T32 GM07215 (to A.B.B.).

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