HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 30, 27282-27287, July 26, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/30/27282    most recent M202881200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tam, C. Articles by Missiakas, D. Search for Related Content PubMed PubMed Citation Articles by Tam, C. Articles by Missiakas, D. Social Bookmarking                      What's this?

Interaction of the Conserved Region 4.2 of ^E with the RseA Anti-sigma Factor^*

Christina Tam, Bruno Collinet, Gary Lau, Satish Raina, and Dominique Missiakas^§

From the Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637 and the  Departement de Biochimie Médicale, Centre Médical Universitaire, 1 Rue Michel Servet, 1211 Geneva 4, Switzerland

Received for publication, March 25, 2002, and in revised form, May 8, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

E^E RNA polymerase transcribes a regulon of folding factors for the bacterial envelope and is induced by physical and chemical stresses. The RseA anti-sigma factor inhibits the activity of E^E RNA polymerase. It is shown here that the N-terminal portion of ^E, residues 1-153, binds core RNA polymerase. RseA interacts with residues 154-191 of ^E, a site that is homologous to region 4, the sigma factor binding site for promoter DNA. Mutations that reduce transcription of E^E RNA polymerase map to ^E residues 178, 181, and 183. Variant ^E proteins with amino acid substitutions at residues 178, 181, or 183 do not associate with RseA. A regulatory mechanism is proposed whereby RseA binds to a C-terminal peptide of ^E and inhibits the transcription of E^E RNA polymerase by blocking promoter recognition.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

^E (RpoE)¹ is a member of the extracytoplasmic function (ECF) subfamily of sigma factors, which transcribes genes that encode protein folding factors in response to extracytoplasmic stress stimuli (1, 2). In Escherichia coli, the unfolding of proteins in the cell envelope appears to be a primary stimulus that activates the ^E-dependent response (1, 3). Previous work identified genes that are transcribed by E^E RNA polymerase. The E^E regulon controls at least two cellular processes, folding of polypeptides in the bacterial envelope and biosynthesis/transport of lipopolysaccharides (4). ^E is encoded by the rpoE gene and in part regulates its own expression, as E^E RNA polymerase transcribes rpoE as well as the downstream genes rseA/B/C.

Conditions that cause unfolding of polypeptides in the envelope signal the E^E response by a mechanism that requires RseA and RseB (5). The genes for these two regulators of E^E are located immediately downstream of rpoE. RseA is a short polypeptide that integrates into the cytoplasmic membrane. The N-terminal cytoplasmic domain of RseA is known to bind ^E, whereas the C-terminal domain of RseA protrudes into the periplasm. The C-terminal domain of RseA interacts with RseB in the periplasm, a compartment that is located between the inner and outer membranes of E. coli (6, 7). RseB binding to RseA increases the affinity of the RseA/RseB complex for ^E. Stresses that cause unfolding of proteins in the bacterial envelope lead to the dissociation of RseA/RseB, thereby reducing the affinity of RseA for ^E.

The domain structure of sigma factors has been probed with trypsin cleavage of the peptide backbone under conditions of limited proteolysis (8). ⁷⁰, the major sigma factor of E. coli RNA polymerase, is a 613-amino acid residue polypeptide with two preferred trypsin cleavage sites, suggesting that it is assembled from folded subdomains (see diagram in Fig. 1). The N-terminal cleavage fragment of ⁷⁰, residues 1-114, has not yet been analyzed in depth. The central domain, ⁷⁰₂ (residues 104-448), is capable of associating with core RNA polymerase in a manner that allows binding of the ⁷⁰₂ holoenzyme to single stranded DNA oligonucleotides that encompass the promoter binding site for ⁷⁰ RNA polymerase. Despite this DNA binding activity, ⁷⁰₂ holoenzyme does not promote transcription in vitro. The C-terminal trypsin fragment of ⁷⁰, ⁷⁰_3,4 (residues 449-613), binds to the coliphage T4 anti-sigma factor AsiA (9-11). The formation of a complex between AsiA with the C-terminal domain 4.2 of ⁷⁰-(551-608) prevents the association of RNA polymerase holoenzyme with 35 promoter sequences and blocks ⁷⁰ RNA polymerase transcription during T4 coliphage infection. Rsd, the anti-sigma factor of E. coli, also binds to region 4.2, suggesting that inactivation of ⁷⁰ may occur by a similar mechanism (12-14). Promoters that are composed of an extended 10 binding site for ⁷⁰ RNA polymerase but lack the canonical 35 sequence can be transcribed by a truncated RNA polymerase, ⁷⁰-(1-529) (15). Although this has not yet been tested experimentally, ⁷⁰-(1-529) is presumably refractory to AsiA- or Rsd-mediated inhibition.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Domain structure of 70 and E. Regions 1, 2, 3, and 4 of E were defined by Lonetto et al. (2) using BLAST searches with 70. HTH indicates the position of a helix-turn-helix motif. 702 (residues 104-448) and 703,4 (residues 449-613) are two fragments obtained by limited trypsin digestion of 70. 702 is capable of associating with core RNA polymerase but does not promote transcription in vitro. 703,4 binds to Rsd and to the coliphage T4 anti-sigma factor, AsiA. This study examines the E domains involved in binding to RNA polymerase core or the anti-sigma factor RseA; the results are summarized in the drawing.

By using limited trypsin digestion and peptide mapping, it is reported here that an N-terminal portion of ^E, residues 1-153, binds core RNA polymerase but not RseA. The remaining portion of ^E, residues 154-191, is homologous to region 4.2, the sigma factor binding site for promoter DNA. In order to establish the biochemical activity of the putative region 4.2, mutations that reduce transcription of E^E RNA polymerase were isolated and mapped to ^E residues 178, 181, and 183. The variant ^E proteins failed to associate with RseA. These findings suggest that binding of RseA to region 4.2 of ^E prevents transcription of E^E RNA polymerase by blocking promoter recognition.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strains and Growth Conditions-- Strains used in this study are listed in Table I. Sequences of primers used in this study can be obtained from the authors upon request. When necessary, Luria Bertani (LB) medium was supplemented with ampicillin (100 µg/ml), kanamycin (50 µg/ml), or tetracycline (15 µg/ml). Induction of _HisRseA^N and RseA^N_His and the various _His^E truncations or mutants was accomplished by addition of arabinose (0.2%) or isopropyl-1-thio--D-galactopyranoside (1 mM) to cultures of E. coli LMG194 or BL21, respectively, harboring the appropriate plasmids. rpoE alleles were transduced along with the linked nadB::Tet marker to the desired background using P1 bacteriophage as described (16).

Cloning Procedures-- The DNA regions corresponding to fragments or mutants of ^E polypeptide were amplified by PCR using appropriate primers and cloned into pBAD-B vector (Invitrogen) using the appropriate restriction sites. Plasmids were verified by DNA sequence analysis. In this manner, the cloned DNA fragments were fused to generate a translational fusion between a 6-histidine tag followed by a cleavable enterokinase site. To improve production yields, the DNA fragments were subcloned into pET-24d vector, using NcoI and EcoRI restriction sites. Such plasmids were transformed in strain BL21, and gene expression was induced by addition of isopropyl-1-thio--D-galactopyranoside.

Protein Purification-- The histidine-tagged N-terminal RseA proteins were purified from cell extracts of E. coli strain LMG194 (pBAD-B-_HisRseA^N; the 6-histidine tag can be removed by enterokinase cleavage) or E. coli strain BL21 (pET-24d-RseA^N_His). Cells of 2L culture were harvested by centrifugation at 3,000 × g for 10 min, suspended in buffer A (50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, pH 8.0), and lysed in a French pressure cell at 14,000 p.s.i. Unbroken cells were removed by centrifugation at 3,000 × g for 10 min, and the supernatant was centrifuged at 100,000 × g for 45 min at 4 °C. RseA^N pelleted with the insoluble material. The protein was recovered from the pellet using buffer A containing 8 M urea. This treatment solubilized the protein that could be recovered after centrifugation at 100,000 × g for 45 min at 4 °C to 90% homogeneity. The supernatant was subjected to affinity chromatography using 1 ml of nickel-NTA resin (Qiagen). The resin was washed with buffer A supplemented with 10 mM imidazole, and RseA^N with a C-terminal histidine tag was eluted using an imidazole gradient. The N-terminal tagged RseA^N protein was recovered by treatment of the beads with enterokinase as indicated by the manufacturer (Invitrogen). Purification of _His^E full-length and the various protein mutants and fragments was performed as described for _His^E (5).

Binding Assays-- For affinity measurements, 50% slurry of nickel-NTA was prepared that contained bound 6-histidine-tagged proteins (RseA^N, wild-type ^E, or mutant ^E or ^E fragments each at 1-2 pmol protein/µl slurry). Increasing concentrations of substrate proteins (only the 5- or 10-fold molar excess are shown in the figures) were added to a 1-ml suspension of 50 µl of nickel-NTA-Sepharose charged with bait protein in buffer A containing 0.2% octylglucoside. Samples were incubated for 2 h at 20 °C and centrifuged at 3,000 × g for 5 min. The supernatant (850 µl) was removed and protein precipitated with 10% trichloroacetic acid. Sediments were washed with acetone, solubilized in 50 µl of 0.1 M Tris-HCl, 4% SDS, pH 7.0, and heated at 95 °C for 5 min. The beads were washed three times with 1 ml of buffer A containing 0.2% octylglucoside. Washes were completed within 10 min, and no significant elution was observed during washes. Samples were separated on 12% SDS-PAGE, electrotransferred to polyvinylidene difluoride membrane and immunoblotted with anti-^E, anti-RseA, or anti-RpoA (the  subunit of core polymerase) antibodies. Immune complexes were detected using a secondary antibody linked to horseradish peroxidase.

Biochemical Assays-- -Galactosidase activity was determined as described previously (16). Modification of sulfhydryl groups using Ellman's reagent (dithionitrobenzoate (DTNB)) was performed at 20 °C using 20 mM Tris-HCl buffer, pH 7.5, containing 0.5 mM EDTA and 8 µM ^E protein in the presence or absence of RseA^N (a 2-160 µM range of concentration was used; only the 10-fold molar excess is shown in Fig. 4). Upon addition of DTNB (250 µM) to a 1-ml reaction volume, formation of 5-thio-2-nitrobenzoate was monitored at 412 nm and quantified using the extinction coefficient value of 14150 M¹cm¹ (17).

Trypsin digestion reactions contained 20 mM Tris-HCl (pH 7.9), 50 mM NaCl, 5% glycerol, 0.1 mM EDTA, 200 pmol of ^E, and L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma) at either a 1:200, 1:400, or 1:800 ratio of trypsin over ^E. The reactions were incubated at 25 °C and stopped by addition of 1 mM phenylmethylsulfonyl fluoride and 10% trichloroacetic acid. Proteins were separated on 15% SDS-PAGE and analyzed by immunoblotting using anti-^E antibodies. Edman degradation and electrospray-ionization mass spectrometry measurements of trypsin digests were performed by the Rockefeller University Protein Sequencing Facility and the Mass Spectrometry Facility at the University of California, Los Angeles.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Trypsin Digestion of ^E-- We sought to probe the domain structure of the E. coli extracellular sigma factor, ^E, by trypsin cleavage. ^E protein was purified from an engineered E. coli strain that overexpressed a His-tagged variant of the sigma factor using affinity chromatography. The N-terminal histidine tag was removed by enterokinase cleavage, causing ^E elution from nickel-NTA-Sepharose. The eluate was subjected to ion exchange chromatography yielding a more than 95% pure preparation of ^E. Purified ^E is soluble and stable at 4 °C. It is capable of binding core RNA polymerase or the RseA anti-sigma factor (see below). These results suggested that our purified ^E was correctly folded and functional. Incubation of purified ^E with dilute amounts of trypsin caused peptide cuts at many different sites, generating a large spectrum of cleavage fragments. A 10-kDa fragment of ^E appeared to be resistant to cleavage by dilute amounts of trypsin (Fig. 2). Nevertheless, an increase in the molar ratio of trypsin to ^E to 1/200 caused cleavage of the entire polypeptide into fragments of small sizes that eluded further analysis on SDS-PAGE. Thus, in contrast to the trypsin-resistant ⁷⁰, ^E seems sensitive to protease cleavage, suggesting a folded structure that is less compact and more accessible to protease.

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Trypsin digestion of E. Purified E was incubated with trypsin for 30 and 60 min at 200, 400, or 800 molar excess E over trypsin (lanes 3-8). Proteolysis was quenched by the addition of phenylmethylsulfonyl fluoride and precipitation with trichloroacetic acid. Protein was separated on 15% SDS-PAGE and analyzed by Coomassie Blue staining. Electrospray-ionization mass spectrometry revealed an average compound mass of 10506.0 for the10-kDa cleavage fragment. Edman degradation revealed the presence of the N-terminal methionine as well as the predicted peptide sequence, indicating that trypsin had cleaved the C-terminal of E and that the 10-kDa peptide comprises E residues 1-91. Prestained molecular weight markers were separated in lanes 1 and 9; the numbers indicate the molecular mass in kDa. Purified undigested E was separated in lane 2 (mock).

Electrospray-ionization mass spectrometry and Edman degradation were used to characterize the 10-kDa fragment of ^E that resisted trypsin cleavage at dilute or intermediate enzyme concentrations. The 10-kDa fragment corresponded to amino acid residues 1-91 of ^E. This portion of ^E carries a peptide with sequence homology to region 1 and region 2 of ⁷⁰ (Fig. 1). Region 2 of ⁷⁰ contains four functional subdomains, designated 2.1, 2.2, 2.3, and 2.4. Homologous subdomains were also identified within ^E using BLAST homology searches (Fig. 1). Subdomain 2.1 represents the binding region for RNA polymerase core enzyme, whereas subdomain 2.3 contributes to DNA melting. The subdomain 2.4 is involved in promoter recognition in the 10 nucleotides region upstream of the transcriptional start site. The N-terminal trypsin-resistant fragment of ^E (residues 1-91) binds neither to core RNA polymerase nor to RseA (see below). Together these results indicate that, unlike ⁷⁰, trypsin cleavage of ^E does not separate folded domains with distinct function.

A Subdomain of ^E Binds Core RNA Polymerase-- We sought to map the binding sites of ^E for both RNA polymerase core enzyme and the anti-sigma factor RseA. N- and C-terminal truncations of ^E were generated by PCR amplification using specific primers and rpoE template DNA (rpoE encodes ^E). _His^E (amino acids 1-191), _His^E_1-91, _His^E_1-116, _His^E_1-137, _His^E_1-153, and _His^E_95-191 are rpoE variants of variable lengths bearing an N-terminal histidine tag. The subscript numbers indicate the RpoE amino acid positions at the beginning and the end of each fragment, respectively. His-tagged ^E variants were purified by affinity chromatography, and their ability to interact with either RseA^N or core RNA polymerase was tested in vitro.

Aliquots of nickel-NTA-Sepharose precharged with ^E protein were dispensed as a 50% slurry into buffer containing 10-fold excess of purified E. coli core RNA polymerase. _His^E-₂' binding was measured as the amount of RNA polymerase  subunit that sedimented during 4,000 × g centrifugation with nickel-NTA-Sepharose. Full-length RpoE, _His^E, and the C-terminal truncated variant _His^E_1-153, caused co-sedimentation of core RNA polymerase with ^E-charged resin (Fig. 3A). These data suggest that RpoE residues 154-191, a peptide sequence homologous to the ⁷⁰ 4.2 domain involved in binding the 35 region of promoter DNA, is not required for ^E binding to RNA polymerase. As a control, mock-charged nickel-NTA-Sepharose did not associate with RNA polymerase. N-terminal truncation of the presumed binding region for core RNA polymerase, domains 1.2, 2.1, 2.2, and 2.3, indeed prevented the variant ^E protein (^E_95-191) to capture the  subunit of RNA polymerase. A similar result was observed for all C-terminal truncations tested, as nickel-NTA-Sepharose charged with _His^E_1-91, _His^E_1-116, or _His^E_1-137 failed to promote co-sedimentation of RNA polymerase (Fig. 3A).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Interaction of E fragments with core polymerase and RseAN. Binding of core RNA polymerase or RseAN to the E fragments was assessed in a co-sedimentation assay using nickel-NTA-Sepharose. Sepharose resin was charged with histidine-tagged polypeptides and used as bait to capture proteins in solution (50% slurry of beads). Proteins that are bound to the resin were collected by slow speed centrifugation, washed three times, and eluted with imidazole. Both bound and unbound proteins were precipitated with trichloroacetic acid, washed in acetone, and analyzed on 12% SDS-PAGE followed by immunoblotting with antibodies directed against RpoA (the  subunit of RNA polymerase) (A), RseAN (B), or E (C). Full-length E spans residues 1-191. The fragments E1-91, E1-116, E1-137, E1-153, and E95-191 are depicted as 1-91, 1-116, 1-137, 1-153, and 95-191. E proteins were used at a final concentration of 1 pmol/µl resin and incubated with purified core RNA polymerase (10-fold excess) for 2 h at room temperature (A) or 5-fold excess of purified RseAN for 2 h at room temperature (B). As RseAN did not co-sediment with histidine-tagged E fragments (B), the reciprocal experiment was performed (C) by charging nickel-NTA resin with RseAHis (2 pmol/µl resin) and incubating RseAN with 10-fold excess of E.

RseA is a type II membrane protein with an N-terminal ^E-binding domain that resides in the cytoplasm and a C-terminal RseB-binding domain within the periplasm. RseA^N is a truncated variant containing an N-terminal His tag and a C-terminal truncation to the first residue of the membrane-spanning domain of wild-type RseA. RseA^N was purified from the cytoplasm of E. coli by affinity chromatography and its N-terminal His tag removed by cleavage using enterokinase. Purified RseA^N was added to Ni-NTA-Sepharose pre-equilibrated with _His^E fragments and sigma factor binding was measured by immunoblotting as the amount of RseA^N that sedimented with the Sepharose beads during 4,000 × g centrifugation. Only full-length RpoE, _His^E, but not _His^E_1-91, _His^E_1-116, _His^E_1-137, or _His^E_1-153, caused co-sedimentation of RseA^N with the precharged resin (Fig. 3B). To confirm these findings, a reciprocal experiment was conducted (Fig. 3C). Ni-NTA-Sepharose charged with _HisRseA^N was incubated with the ^E and its variants ^E_1-91, ^E_1-116, ^E_1-137, ^E_1-153, or ^E_95-191. The His tag of each variants was removed using enterokinase, and proteins were purified by ion exchange chromatography. Control experiments showed that the purified ^E variants (^E_1-91, ^E_1-116, ^E_1-137, ^E_1-153, or ^E_95-191) did not co-sediment with mock-charged nickel-NTA-Sepharose beads (Fig. 3C). Our assay could detect the binding of RseA^N to full-length ^E. All C-terminal truncations of ^E abolished the association with RseA^N, suggesting that C-terminal ^E sequences are required for RseA binding. Failure to observe binding between ^E_95-191 and RseA^N can be attributed to a lack of structure or stability of ^E_95-191. Indeed when RseA^N was overproduced along with ^E_95-191 in the same cell, a soluble ^E_95-191-RseA^N complex could be extracted from total cell extracts (data not shown).

Accessibility of Cysteine 165 of ^E to Ellman's Reagent-- Amino acids 1-153 of ^E polypeptide (fragment ^E_1-153) are sufficient to promote interaction between the sigma factor and core RNA polymerase, but fail to associate with RseA^N. We entertained the possibility that interaction with RseA occurs at a C-terminal portion of ^E. rpoE codon 165 encodes cysteine, a sulfhydryl-containing amino acid, within the C-terminal peptide sequence (amino acids 154-191). Cysteine 165, Cys¹⁶⁵, is the only cysteine residue within ^E. Reaction of Ellman's reagent (DTNB) with sulfhydryl residues results in the formation of 5-thio-2-nitrobenzoate, a product that absorbs light at 412 nm. Cys¹⁶⁵ is accessible to Ellman's reagent because incubation of ^E with this compound led to the rapid generation (less than 30 s) of the product with absorbance at 412 nm (Fig. 4). The extent of release of thionitrobenzoate did not yield a molar ratio of DTNB over ^E, as the amount of reacting thiol was 12 µM and the calculated ^E concentration was 8 µM. Either the preparation of ^E was contaminated with thiol-containing proteins or the concentration of ^E, deduced from the calculated molar extinction coefficient _278 nm 15,400 M¹ cm¹, was underestimated. Nonetheless, further incubation of this reaction did not increase the absorbance at 412 nm, a finding that is consistent with the notion that all available sulfhydryl moieties had been modified within 30 s. Incubation of Ellman's reagent with complexes formed from equimolar amounts of ^E and RseA^N generated only a modest amount of absorbance at 412 nm. Further, the absorbance at 412 nm during this experiment increased slowly with a steady rate while incubating for 800 s (Fig. 4). These data are consistent with a model in which the binding of RseA to the C-terminal portion of ^E blocks the access of Ellman's reagent to Cys¹⁶⁵. Further, the slow increase in absorbance is likely caused by the dissociation of ^E-RseA^N, thereby liberating the sulfhydryl at position 165 for modification with Ellman's reagent. RseA^N does not contain cysteine residues, and incubation of RseA^N alone with Ellman's reagent did not yield a significant absorbance at 412 nm (data not shown). Covalent modification of ^E with Ellman's reagent does not prevent the association of the sigma factor with either RseA^N or core RNA polymerase, suggesting that Cys¹⁶⁵ is not an essential residue for the formation of the ^E-RseA^N complex (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Accessibility of C165 of E to Ellman's reagent in the presence or absence of RseAN. Ellman's reagent (DTNB) was added at a final concentration of 250 µM to E (8 µM) in the absence or presence of RseAN (8 µM). The total volume of the reaction was 1 ml. Change in absorbance was recorded for 800 s at 412 nm using a Varian spectrophotometer thermostatted at 20 °C.

RseA Interacts with Region 4.2 of ^E Polypeptide-- Cys¹⁶⁵ is located in a region of ^E that is homologous to the 4.2 domain of ⁷⁰ (Fig. 1). Region 4.2 of ⁷⁰ contains a putative helix-turn-helix DNA-binding motif that interacts with the 35 element of ⁷⁰-dependent promoters. We assumed that mutations within the 4.2 region of ^E may likely display defects in the transcription at E^E-dependent promoters. To test this prediction, we sought to isolate mutations that decrease the transcription of E^E-dependent promoters. A strain carrying the nadB::tet allele was mutagenized with hydroxylamine (18). The nadB gene is linked to the rpoE operon. Bacteriophage P1 lysates of mutated cultures were transduced into E. coli MC4100 carrying the rpoEP2-lacZ transcriptional fusion. Mutations linked to the Tet^R marker were selected for by plating on agar containing 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal; 20 µg/ml) and tetracycline. Light blue Tet^R transductants were retained. Only mutations that co-transduced with tet^R at a frequency greater than 90% linked to the Tet^R marker and that conferred temperature-sensitive growth above 37 °C were retained. The location of rpoE mutations was determined by DNA sequencing.

Three of the isolated mutations changed the specificity of single codons within the 4.2 domain of ^E: R178G, I181A, and V185A. Each of the three mutations decreased transcriptional activity of ^E RNA polymerase at multiple promoters (htrA-lacZ, rpoEP2-lacZ and rpoHP3-lacZ), suggesting that the regulatory defects were neither allele- nor promoter-specific (Fig. 5).

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Transcriptional activity of various E mutants in vivo. To test the activity of the E mutants in vivo, the rpoER178G, rpoEI181A, and rpoEV185A alleles were transduced into E. coli strain MC4100 carrying chromosomal insertions of the reporter genes rpoHP3-lacZ, rpoEP2-lacZ, or htrA-lacZ, respectively. Bacteria were grown overnight at 30 °C, diluted 1:100, and allowed to reach A595 nm between 0.5 and 0.7. -Galactosidase activity measurements were performed in duplicate and calculated in Miller units (16). Data represent the average of at least three independent experiments.

We wondered whether rpoE mutations that affect promoter selection and/or transcriptional activity by ^E RNA polymerase caused a simultaneous effect on the binding of ^E to RseA. This possibility was tested in a biochemical experiment. Three mutant rpoE alleles were amplified by polymerase chain reaction, and the corresponding genes were cloned in the pET expression vector. The mutant proteins, _His^E_R178G, _His^E_I181A, and _His^E_V185A, were purified by affinity chromatography on nickel-NTA-Sepharose. When measured with a co-sedimentation assay, _His^E_R178G, _His^E_I181A, and _His^E_V185A bound to purified RNA core polymerase in a manner that was indistinguishable from the binding of wild-type ^E. Fig. 6 shows the results of these experiments for wild-type ^E and ^E_R178G. To measure binding of ^E to RseA, nickel-NTA-Sepharose beads were charged with RseA^N and incubated with enterokinase-cleaved ^E protein. Although co-sedimentation of wild-type ^E and RseA^N was observed, neither ^E_R178G nor ^E_I181A or ^E_V185A interacted with RseA^N. Fig. 6 shows the results of the experiments for wild-type ^E and ^E_R178G. Alanine substitution of Cys¹⁶⁵, the sulfhydryl residue, did not affect the in vitro binding of ^E to core polymerase or to RseA^N (data not shown).

View larger version (38K): [in this window] [in a new window]   Fig. 6.   Interaction of E and ER178G with RseAN and core RNA polymerase. A, nickel-NTA resin was charged with 1 µM RseAN and incubated with increasing concentrations of E or ER178G. B, nickel-NTA resin was charged with 1 µM HisE or HisER178G and incubated with increasing concentrations of core RNA polymerase. Bound proteins were collected by low speed centrifugation, washed three times with the resin, and eluted with imidazole. Protein was precipitated with trichloroacetic acid, washed in acetone, and subjected to 12% SDS-PAGE and immunoblotting with antibodies raised against E (-E) or the  subunit of RNA polymerase (-RpoA).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

^E is a member of the ⁷⁰ family of proteins. These sigma factors are modular proteins, consisting of four conserved regions and their subregions (Fig. 1; reviewed in Ref. 19). Regions 2.4 and 4.2 encode two DNA binding determinants that allow for recognition of the conserved 10 and 35 regions of promoters, respectively. Region 1.1 is an autoinhibitory domain that masks the DNA binding determinants of ⁷⁰ when the transcription factor is not associated with RNA polymerase (20). Interaction with core polymerase relieves region 1.1-mediated autoinhibition (20) and reorients regions 3.1-4.2 of ⁷⁰ relative to the central 1.2-2.4 domains. These movements permit the selective recognition of 10 region promoter sequences by regions 2.3-2.4 (21). Although region 1.1 is present in many primary  factors (⁷⁰), this domain is absent in ^E. Mutational changes in regions 2, 3, and 4 of ⁷⁰ have been shown to decrease binding to RNA polymerase, and it has been proposed that putative contact sites exist between regions 2, 3, 4, and the ₂' core (22).

We have used limited trypsin digestion to probe the domain organization of ^E. One fragment resisted digestion by dilute amounts of trypsin and contained region 1.2 as well as region 2:^E amino acids 1-91. These data correlate well with preliminary NMR experiments in which ^E residues 92-191, unlike amino acids 1-91, generated little resonance (data not shown). Thus, the C-terminal part of ^E (residues 92-191) does not assume a compact fold. The N-terminal peptide, ^E_1-91, was cloned, expressed, and purified by affinity chromatography. Purified ^E_1-91 did not bind RseA^N or core polymerase. The latter observation was a surprise to us, as fragment ⁷⁰₂, comprising region 1.2 and region 2 of ⁷⁰ (39 kDa), is known to bind to core RNA polymerase in a manner that competes with the binding of wild-type ⁷⁰ (8). Although our measurements for binding of ^E fragments to core polymerase employed an assay similar to that of the ⁷⁰ studies, pronounced differences were observed (8). Although all three fragments, ^E_1-116, ^E_1-137, and ^E_1-153, comprise region 1.2, only the largest fragment, ^E_1-153, was capable of binding to core polymerase.

The fact that RseA did not bind to ^E_1-153, together with the observation that region 4.2 is located in the peptide sequence 153-191, suggested that RseA may bind region 4.2 of ^E. A direct demonstration of this hypothesis could not be achieved as ^E_95-191 did not co-sediment with RseA^N. One explanation for the negative result is a lack of structure of the C-terminal domain of ^E. Is it possible that RseA does bind to region 4.2 but only if this domain is tethered to the remainder of ^E? This notion is corroborated by several observations. First, ^E_95-191 is unstable and rapidly forms aggregates that can be sedimented by centrifugation. Second, binding of RseA to ^E masks Cys¹⁶⁵ for modification with Ellman's reagent, as if RseA occupies residues 153-191 (region 4.2). Third, region 4.2 was also characterized by isolating ^E variants that fail to promote ₂' ^E-mediated transcription of ^E-dependent promoters. Three amino acid substitutions mapped to region 4.2: R178G, I181A, and V185A. When tested in a biochemical experiment, all three ^E variants failed to bind RseA although the association with core RNA polymerase was similar to that of wild-type ^E. Together these data suggest that RseA indeed binds to region 4.2 of ^E.

The activity of several sigma factors is regulated by a cognate anti-sigma factor. Many pairs of sigma/anti-sigma factors have been identified, and their biological roles have been characterized (9, 23). Recently, the structural features of some anti-sigma factors and the binding sites for the cognate sigma factors have been elucidated (10, 11, 14, 24, 25). The two anti-sigma factors of ⁷⁰, AsiA and Rsd, are about 10 kDa of mass. AsiA is encoded by the bacteriophage T4, and its association with the ⁷⁰ subunit of the E. coli RNA polymerase is one of the principal events controlling transcription of host cells and of the T4 genome. The recognition site of AsiA on ⁷⁰ has been mapped using NMR and alanine-scanning mutagenesis (10, 11). These studies showed that the highly conserved region 4.2 (and maybe 4.1) constitutes the AsiA-binding domain. Mutations that affect AsiA binding to ⁷⁰ are clustered in a stretch of 5 residues within the C-terminal half of region 4.2 (KAL⁵⁹⁵RK). These residues belong to a loop of charged residues (KAL⁵⁹⁵RKLRHPS) where Lys⁵⁹³, Arg⁵⁹⁶, Lys⁵⁹⁷, Arg⁵⁹⁹, and His⁶⁰⁰ contribute to the interaction with protein activators of transcription rather than directly to ⁷⁰ binding of DNA (26, 27). Modeling of residues 551 to 613 of ⁷⁰ on the structure of NarL and 434 Cro suggests that all of the charged residues are surfaced exposed in the HTH motif (26). Strikingly, RseA binding is dramatically affected by mutations clustered in the C-terminal half of region 4.2 of ^E (R178G, I181A, and V185A). Sequence alignment between ^E and ⁷⁰ shows that this region of ^E (RAR¹⁷⁸EAI¹⁸¹DNKV¹⁸⁵) corresponds to the stretch of charged residues described above for ⁷⁰ (KAL⁵⁹⁵RKLRHPS). Within ^E these residues are also important for interaction with the DNA as alterations of Arg¹⁷⁸, Ile¹⁸¹, and Val¹⁸⁵ lead to decreased ^E activity in vivo. The recognition site of Rsd, a second anti-⁷⁰ factor produced by E. coli during stationary phase (13), has also been mapped to region 4 of ⁷⁰ (12, 14). Alanine substitutions at two positions flanking arginine 596 (Leu⁵⁹⁵ and Leu⁵⁹⁸) disrupt the association of Rsd with ⁷⁰ in vitro (14). Hence, the same region of ⁷⁰ that is targeted for recognition by two unrelated anti-sigma factors appears to be important for the ^E-RseA interaction.

	ACKNOWLEDGEMENTS

We thank K. Faull (University of California, Los Angeles) for mass spectrometry measurements and O. Schneewind (University of Chicago) for critical review of this manuscript.

	FOOTNOTES

^* This work was supported by United States Public Health Service Grant GM58266.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.

^§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, The University of Chicago, 920 East 58th St., Chicago IL 6063. Tel.: 773-834-8161; E-mail: dmissiak@bsd.uchicago.edu.

Published, JBC Papers in Press, May 16, 2002, DOI 10.1074/jbc.M202881200

	ABBREVIATIONS

The abbreviations used are: RpoE or ^E, sigma E transcription factor; E or ₂', core RNA polymerase; E^E or ₂' ^E, holoenzyme complexed to sigma E; Rse, regulator of ^E; rpoER178G, rpoEI181A, and rpoEV185A, alleles of rpoE encoding mutants of ^E with severely impaired transcriptional activity; _HisRseA^N and RseA^N_His, N-terminal domain of RseA fused to a 6-histidine tag at the N terminus or C terminus, respectively; NTA, nitrilotriacetic acid; DTNB, dithionitrobenzoate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				S. Mahren and V. Braun The FecI Extracytoplasmic-Function Sigma Factor of Escherichia coli Interacts with the {beta}' Subunit of RNA Polymerase J. Bacteriol., March 15, 2003; 185(6): 1796 - 1802. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/30/27282    most recent M202881200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tam, C. Articles by Missiakas, D. Search for Related Content PubMed PubMed Citation Articles by Tam, C. Articles by Missiakas, D. Social Bookmarking                      What's this?