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Journal of Bacteriology, May 2001, p. 2952-2956, Vol. 183, No. 9
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.9.2952-2956.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Mapping of the Rsd Contact Site on the Sigma 70 Subunit of Escherichia coli RNA Polymerase

Miki Jishage, Dipak Dasgupta, and Akira Ishihama^*

National Institute of Genetics, Department of Molecular Genetics, Mishima, Shizuoka 411-8540, Japan

Received 21 December 2000/Accepted 20 February 2001

	ABSTRACT

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Rsd (regulator of sigma D) is an anti-sigma factor for the Escherichia coli RNA polymerase ⁷⁰ subunit. The contact site of Rsd on ⁷⁰ was analyzed after mapping of the contact-dependent cleavage sites by Rsd-tethered iron-p-bromoacetamidobenzyl EDTA and by analysis of the complex formation between Ala-substituted ⁷⁰ and Rsd. Results indicate that the Rsd contact site is located downstream of the promoter 35 recognition helix-turn-helix motif within region 4, overlapping with the regions involved in interaction with both core enzyme and ⁷⁰ contact transcription factors.

	TEXT

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The survival of bacterial cells in various environments depends on their abilities to sense the external conditions and adapt their internal metabolic systems by turning on and off the expression of specific genes (for reviews, see references 12 and 21). For quick change of the global gene expression pattern in response to sudden environmental changes, bacteria carry modulation systems for the specificity and activity of transcription apparatus. The transcription apparatus of Escherichia coli is composed of the RNA polymerase core enzyme (subunit composition, ₂') with the catalytic activity of RNA polymerization and one of seven species of the  subunit with the promoter recognition activity (reviewed in references 14, 15, 19, and 22). The major  subunit, ⁷⁰, is responsible for transcription of most genes expressed during steady-state cell growth under laboratory culture conditions. The other six species of the  subunit are required only during certain growth stages or under specific stress conditions. In agreement with their functional roles, the levels of these alternative  subunits vary depending on the cell growth conditions (25, 26, 42), and all the  subunits compete with each other for binding to a fixed amount of the core enzyme (41). In addition to the level control, the activities of at least some E. coli  subunits are under a control system in which the unused  subunits are stored in inactive forms by forming complexes with another set of proteins, often designated as anti- factors, with the regulatory activity of  functions (for reviews see references 18 and 21).

Subunit ^F is involved in transcription of the genes needed for flagellum formation and chemotaxis. The flgM gene product is an anti-^F factor that acts by directly binding to ^F and thereby preventing its interaction with the core RNA polymerase (33). Subunit ^E is a member of the ECF family of  subunits for transcription of the genes related to extracytoplasmic functions (39) as well as those required for high temperature survival or thermotolerance (9). The ^E activity is regulated by the rseA (regulator of sigma E) gene product or anti-^E factor, which is associated with the inner membrane and inhibits the activity of ^E by directly interacting with ^E (7, 43). FecI also belongs to the ECF  family and is involved in transcription activation of the ferric-citrate transport genes (fec) (1). Genetic studies revealed that FecR, an inner membrane protein, negatively regulates the activity of the FecI  subunit (49). FlgM, RseA, and FecR are classified as members of anti-sigma factors for ^F, ^E, and ^FecI, respectively. A heat shock protein, DnaK, can be an anti- factor for the heat shock ^H subunit (18), which is induced following heat shock and is involved in transcription of the genes encoding heat shock proteins, including DnaK, DnaJ, and GrpE (13). After returning from the transient adaptation period to heat shock to steady-state growth at high temperatures, unused ^H becomes stored as DnaJ-DnaK-^H complexes (38), which are dissociated by the action of GrpE to release ^H for reuse or for degradation by HflB (FtsH) protease (10).

Recently we discovered a novel E. coli protein, referred to as Rsd (regulator of sigma D), which forms a complex with ⁷⁰, the major ⁷⁰ subunit for growth-related gene transcription, and prevents its function (24). Purified Rsd protein formed complexes in vitro with ⁷⁰ but not with other  subunits and inhibited transcription in vitro by the holoenzyme containing ⁷⁰ to various extents, depending on the promoters used (24). Since Rsd is induced in the stationary phase of cell growth, where ⁷⁰ is not used, we proposed that Rsd is an anti- factor for the major ⁷⁰ subunit for storage in the stationary phase. In E. coli mutants lacking the rsd gene, the expression of ⁷⁰-dependent genes increases while transient overproduction of Rsd leads to a reduction in ⁷⁰-dependent gene expression (23). Based on these results, taken together, we proposed that Rsd is an anti-sigma factor for the ⁷⁰ subunit.

Cleavage sites of ⁷⁰ by Rsd-tethered FeBABE. Previously we estimated the contact site of Rsd on ⁷⁰ to be downstream from residue 500, including regions 3.2, 4.1, and 4.2, after analysis of complex formation between Rsd and ⁷⁰ fragments (24). For detailed mapping of the contact site of Rsd on the ⁷⁰ subunit, we employed the contact-dependent cleavage of target proteins by FeBABE (iron-p-bromoacetamidobenzyl EDTA)-conjugated pairing proteins (6, 20, 22). In this study, FeBABE was tethered to Rsd at all possible Lys residues by using 2-iminothiolane, which links between Lys and FeBABE (48). For detection of the cleavage sites on ⁷⁰, a protein kinase tag sequence was added at either its N or C terminus and the tag was phosphorylated using [-³²P]ATP and protein kinase A. Mixtures of a fixed amount of ³²P-labeled ⁷⁰ and increasing amounts of FeBABE-tethered Rsd were incubated for 10 min at 37°C to form binary complexes and then were subjected to cleavage reaction by adding ascorbate and H₂O₂. The reaction mixtures were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Fig. 1 shows a tracing of the SDS-PAGE pattern of the cleavage products. As size markers, the same ³²P-labeled ⁷⁰ was treated with CNBr, which induces cleavage at Met residues. Although several nonspecific cleavage products were generated by the addition of H₂O₂ and ascorbate even in the absence of FeBABE-tethered Rsd, the specific cleavage products increased concomitantly with the increase in Rsd-FeBABE addition. At least three such bands, cleaved at sites 1a, 1b, and 2, were identified, each migrating close to the C-terminal CNBr fragment (471/475-613, 488/490-613, or 562/568-613, respectively). Thus we concluded that the FeBABE tethered on the surface of ⁷⁰-bound Rsd approached near the ⁷⁰ segment between residues 471 and 568. The cleavage sites 1a and 1b are located within ⁷⁰ region 3 while cleavage site 2 is within region 4 (see Fig. 3). The result, however, does not immediately indicate the location of the Rsd contact site on these regions because the spacer length between the BABE-tethered Lys and the BABE-associated Fe is about 18 Å (20, 48).

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Cleavage of 70 by Rsd-tethered FeBABE. Purified Rsd was conjugated with FeBABE (Dojin, Kumamoto, Japan) in the presence of 2-iminothiolane according to Traviglia et al. (48), while 70 with a PK tag at the C terminus was labeled in vitro with 32P with protein kinase A. Mixtures of a fixed amount of 32P-labeled 70 (final concentration, 250 nM) and the indicated amounts of FeBABE-tethered Rsd were incubated for 30 min at 37°C and then were subjected to contact-dependent protein cleavage reaction by adding ascorbate and H2O2 followed by SDS-PAGE. CNBr-treated 32P-70 was run on the same gel as size markers. The gel was exposed to an imaging plate, and the plate was analyzed with the BAS 2000 Image Analyzer (Fuji, Tokyo, Japan). Migration is from left to right. The migration positions of CNBr fragments are indicated at the bottom.

The cleavage reaction by Rsd-tethered FeBABE was also performed for all six alternative subunits, ^N, ^S, ^H, ^F, ^E, and ^FecI, but none of them were cleaved even after the addition of excess amounts of Rsd-FeBABE (data not shown). These observations confirm our previous finding that Rsd specifically interacts only with the ⁷⁰ subunit and not with the other  subunits (24).

Rsd-binding activity of Ala-substituted ⁷⁰ mutants. For detailed mapping of the contact site on ⁷⁰ with Rsd, we next tested the complex formation in vitro between Rsd and Ala-substituted ⁷⁰ subunits. The library of Ala-substituted ⁷⁰ was constructed and used for mapping the ⁷⁰ contact sites with the core enzyme (46) or with ⁷⁰ contact transcription factors CRP and FNR (40). The mutant ⁷⁰ subunits with a glutathione S-transferase (GST) tag fused at the N termini were overproduced and were purified to near homogeneity. The GST-tagged ⁷⁰ subunits were mixed with purified Rsd, and the complexes formed were recovered using glutathine-conjugated agarose beads. This GST pull-down assay indicated that two ⁷⁰ mutants with Ala substitutions at residues 595 and 598 were defective in binding to Rsd (Fig. 2A), indicating that the segment of ⁷⁰ including L595 and L598 is involved in molecular interaction with Rsd. The major determinant of core enzyme binding on ⁷⁰ is located in region 2.1 (37). L598 in region 4.2 also participates, at least in part, in binding of the core enzyme (46). The corresponding region of ³² is also involved in core enzyme binding (27). In the case of core enzyme binding, multiple sites on the  subunits are involved and thus a single mutation is often not so critical for overall functions of the  subunits. In fact, under the assay conditions employed, the binding of L598A mutant ⁷⁰ with the core enzyme is stronger than that with the Rsd protein (Fig. 2A).

View larger version (63K): [in this window] [in a new window]   FIG. 2.   Identification of the Rsd contact site on the 70 subunit. (A) GST-70 Ala-substituted mutants were overexpressed and purified to near homogeneity. Each GST-70 mutant was mixed with an equal amount of Rsd, and after incubation for 5 min at 37°C, GST-70-Rsd complexes were isolated by the GST pull-down assay using glutathione-Sepharose beads. The bead-bound proteins were eluted with 50 mM glutathione and were analyzed by SDS-PAGE. The gel was subjected to immunoblotting against anti-Rsd, anti-70, or anti-' antibodies. (B) Activity of the Ala-substituted mutant 70 subunits. In vitro transcription was carried out under the standard reaction conditions (28) using 1 pmol each of the GST-70 mutants, 1 pmol of the core enzyme, and 1 pmol of either lacUV5 or extended 10 promoter DNA fragment (31, 32).

To analyze the role of these residues in the intrinsic ⁷⁰ function of promoting transcription initiation, holoenzymes were reconstituted from each of the Ala-substituted mutant ⁷⁰ subunits and the -free core enzyme and were used for in vitro transcription. The lacUV5-directed transcription was significantly reduced for only the same two mutants, L595A and L598A, which are required for Rsd interaction (Fig. 2B). Thus, the sites required for Rsd interaction are also critical for expression of the intrinsic ⁷⁰ activities, presumably at the step of core enzyme binding (46). The influence of Ala substitution at residues downstream of the 35 recognition helix-turn-helix (HTH) motif of ⁷⁰ was also observed in RNA I promoter-directed transcription in vitro (40). The reduction of ⁷⁰ activity for Ala-substituted mutant ⁷⁰ was, however, not observed when transcription was carried out using the extended 10 promoter (Fig. 2B), which is active in directing transcription even in the absence of 35 promoter -⁷⁰ region 4 interactions (2, 4, 32).

Rsd contact site on the ⁷⁰ subunit. FeBABE cleavage experiments indicate the close location of Rsd near the ⁷⁰ segment between residues 471 and 568, including regions 3.1, 3.2, and 4.1 (see Fig. 1), while the mutant studies using an Ala-substituted ⁷⁰ library indicate that the Rsd contact site is downstream from region 4.2 (see Fig. 2). Since the reactive Fe³⁺ is located 18 Å apart from the Lys residue tethered with FeBABE with the use of a 2-iminothiolane linker (20, 48), it is unlikely that the region between 471 and 568 is the direct target of Rsd binding, but instead the Rsd-binding site includes the residues L595 and L598 identified by Ala scanning. The FeEDTA moiety of the FeBABE tethered at this region may be located close to regions 3 and 4.1, where the cleavage sites were identified (see Fig. 1). Thus we conclude that the direct contact site of Rsd is located on region 4 of the ⁷⁰ subunit downstream of the HTH motif of region 4.2 (Fig. 3), which is involved in recognition of the promoter 35 sequence (11, 47). The 35 contact is, however, not essential for promoter complex formation when the contact of ⁷⁰ region 2 with the promoter 10 sequence alone is strong enough, as in the case of an extended 10 signal (3, 4, 32).

View larger version (16K): [in this window] [in a new window]   FIG. 3.   The Rsd contact sites on the 70 subunit. The predicted Rsd contact sites are compared with the known functional sites, including the sites involved in the molecular interaction with class II transcription factors. The HTH motif is involved in the promoter 35 recognition (11, 47). The contact sites for class II transcription factors are located upstream or downstream of this motif. For details, see the text.

The ⁷⁰ contact (or class II) transcription factors support the functional interaction of ⁷⁰ with promoters lacking the consensus 35 sequence (19). In these cases, the region upstream or downstream of the 35 contact HTH motif in ⁷⁰ region 4 is involved in interaction with class II factors (19, 45). Deletion mutant ⁷⁰ lacking region 4 is still functional with the extended 10 promoter, which alone has a high affinity to the ⁷⁰ region 2 but is defective in response to CRP (on class II promoters) and PhoB (31). Mutant studies indicate that the contact sites for several class II transcription factors, including  cI (8, 30), PhoB (29), CRP (40), FNR (40), Ada (35, 36), AraC (17, 40), and RhaS (3) are all located upstream or downstream of the HTH promoter 35 recognition motif (Fig. 3). Transcription activation by  cI becomes defective in a region 4 mutation of the ⁷⁰ subunit (30). At class II CRP-dependent promoters, CRP makes three different contacts, one of which, known as the activating region 3 (AR3), interacts with region 4 of the ⁷⁰ subunit (45). The positively charged residues K593, K597, and R599 on ⁷⁰ are required for this interaction. Mutations on these residues also affect the ⁷⁰ response to FNR (40). Most class I (or  contact) factors activate transcription by stabilizing the closed complex, while class II (or ⁷⁰ contact) factors such as  cI activate the isomerization step of transcription initiation (8).

The contact site with Rsd is located on the same surface with those of AraC, CRP, FNR, RhaS, and cI (Fig. 3). Thus, the region downstream from the promoter 35 binding HTH of ⁷⁰ seems to be involved in binding the core enzyme, the anti-sigma factor, and a group of class II transcription factors. The anti-⁷⁰ factor Rsd should compete with both the core enzyme and the class II transcription factors in binding with the ⁷⁰ subunit. Likewise, the contact site of FlgM, the anti-^F factor, has been mapped on region 4 of ^F (34). The phage T4 AsiA protein is an anti-sigma factor against the host E. coli ⁷⁰ subunit to repress host cell gene transcription (5, 44). The contact site for the AsiA protein on ⁷⁰ is located within regions 3 and 4 (16). Thus, all the anti- factors so far analyzed seem to interact with the same  region near the promoter 35 recognition surface.

	ACKNOWLEDGMENTS

We thank Carol Gross for donating the expression library of Ala-substituted ⁷⁰ and for helpful discussion.

This work was supported by Grants-in-Aid from the Ministry of Education, Science, Culture and Sports of Japan and by the CREST fund from the Japan Science and Technology Corporation.

	FOOTNOTES

^* Corresponding author. Mailing address: National Institute of Genetics, Department of Molecular Genetics, Mishima, Shizuoka 411-8540, Japan. Phone: (81) 559-81-6741. Fax: (81) 559-81-6746. E-mail: aishiham{at}lab.nig.ac.jp.

Present address: Cancer Institute, Toshima-ku, Tokyo 170-8455, Japan.

On leave of absence from Biophysics Division, Saha Institute of Nuclear Physics, Calcutta-700 037, India.

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• Sharma, U. K., Chatterji, D. (2008). Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences. J. Bacteriol. 190: 3434-3443 [Abstract] [Full Text]  
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