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A Stationary Phase Protein in Escherichia Coli with Binding Activity to the Major Subunit of RNA Polymerase

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A Stationary Phase Protein in Escherichia Coli with Binding Activity to the Major Subunit of RNA Polymerase Proc. Natl. Acad. Sci. USA Vol. 95, pp. 4953–4958, April 1998 Biochemistry A stationary phase protein in Escherichia coli with binding activity to the major s subunit of RNA polymerase (transcriptase controlystationary phase controlyenzyme activity controlyRNA polymerase storage) MIKI JISHAGE AND AKIRA ISHIHAMA* Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411, Japan Communicated by E. Peter Geiduschek, University of California at San Diego, La Jolla, CA, February 6, 1998 (received for review October 22, 1997) ABSTRACT Switching of the transcription pattern in Esch- factors that contribute to the preferential utilization of sS rather erichia coli during the growth transition from exponential to than s70 in the stationary phase or under stress conditions include stationary phase is accompanied by the replacement of the RNA increases in intracellular concentrations of potassium glutamate, polymerase-associated s70 subunit (sD) with s38 (sS). A fraction trehalose, glycine betaine, and polyphosphate and a decrease in of the s70 subunit in stationary phase cell extracts was found to DNA superhelicity, all of which tend to reduce the activity of Es70 exist as a complex with a novel protein, designated Rsd (Regu- but increase that of EsS (16). lator of sigma D). The intracellular level of Rsd starts to increase In addition, we found in this study that in stationary phase cells, during the transition from growing to stationary phase. The rsd s70 but not other s subunits forms a complex with a hitherto gene was identified at 90 min on the E. coli chromosome. unidentified stationary-specific protein. This protein begins to be Overexpressed and purified Rsd protein formed complexes in synthesized during the transition from exponential growth to vitro with s70 but not with other s subunits, sN, sS, sH, sF, and stationary phase. In vitro transcription studies indicated that the sE. Analysis of proteolytic fragments of s70 indicated that Rsd protein interferes with the engagement of s70 in the transcription binds at or downstream of region 4, the promoter 235 recogni- cycle. After sequencing, the protein was identified as a product of tion domain. The isolated Rsd inhibited transcription in vitro to the unidentified reading frames revealed by genome sequence various extents depending on the promoters used. We propose analysis (17). We propose that this protein, designated Rsd that Rsd is a stationary phase E. coli protein with regulatory (Regulator of sigma D), with s70 binding activity plays a role in s70 activity of the function. controlling the s70 function in stationary phase E. coli. The RNA polymerase of Escherichia coli is composed of core MATERIALS AND METHODS a bb9 enzyme with the subunit structure 2 combined with one of Expression Plasmids for the s Subunits, FlgM and Rsd. To seven different species of s subunit. The core enzyme carries all create plasmids for expression in E. coli of various s subunits in the catalytic functions for RNA polymerization, but a s subunit glutathione S-transferase (GST) fusion form, the respective cod- is required for the proper initiation of transcription at promoters ing sequences were PCR-amplified with the primers described in (1–3). The number of core enzyme molecules in an E. coli cell is Table 1. Each primer includes a unique restriction enzyme site approximately 2,000 (4, 5), whereas there are more than 4,000 (underlined in the sequences shown in Table 1). The sequences genes on the E. coli chromosome to be transcribed at widely of the PCR-amplified fragments were confirmed by the varying rates (5, 6). The s subunit required for transcription of dideoxynucleotide method. The PCR-generated DNA fragments most of the genes expressed in the exponential growth phase is were treated with the appropriate restriction enzymes and cloned s70, the product of the rpoD gene (1, 2). Its concentration during into the corresponding sites of the pGEX5x-1 vector (Pharmacia) exponential phase is about 700 molecules per cell, as measured by to generate pGEXD, pGEXN, pGEXS, pGEXH, pGEXF, immunoprecipitation of radiolabeled cell extracts (7) or Western pGEXM, and pGEXRD for expression of GST fusion proteins of blot analysis with specific antibodies (8, 9). In growing cells, at any s70, sN (s54), sS (s38), sN (s32), sF (s28), FlgM (anti-sF), and Rsd, given moment, about two-thirds (about 1,300–1,400 molecules) respectively. The rsd gene was also inserted into pET21a (Nova- of the core enzyme is actively involved in elongation of tran- gen) between the NdeI and XhoI sites to generate pET-Rsd, for scripts. In this state, the core enzyme is bound to DNA but not expression of His -tagged Rsd protein. s 6 associated with . Accordingly most of the remaining core Purification and Analysis of s-Associated Proteins. To iden- enzyme (about 600–700 molecules) is likely to be associated with tify s-associated proteins, an E. coli W3110 type A strain (18) s70 (5, 10). containing pGEXD, pGEXN, pGEXS, or pGEXF was grown in s38 sS Upon entry into stationary phase, or (the product of the Luria–Bertani (LB) medium containing ampicillin (100 mgyml) rpoS gene) begins to be produced and allows core polymerase to at 37°C until stationary phase (3–4 h after cessation of cell recognize and transcribe the genes required for stationary phase growth). Cell lysates were prepared essentially as described sS survival (11–14). The level of increases to as much as about previously (19) and applied onto a glutathione–Sepharose col- s70 250–300 molecules per cell (about 30–35% the level of ) (8, 9). umn (Pharmacia) previously equilibrated with PBS. The column s70 Meanwhile the level of stays constant, even though the was washed with 103 bed volumes of PBS and eluted with s70 frequency of transcription of genes under the control of glutathione-containing buffer (50 mM glutathioney100 mM sS decreases by more than 10-fold (5). The synthesis of is also TriszHCl, pH 8.0y120 mM NaCl). Aliquots of the pooled eluate induced on exposure of growing cells to certain stresses such as fractions were fractionated by electrophoresis on 7.5, 10, and high osmolarity or high temperature, and it plays a role in 13.5% polyacrylamide gels in the presence of SDS. Proteins were transcription of some stress-response genes (14, 15). Known transferred onto PVDF membranes (Nippon Genetics) and stained with Coomassie brilliant blue. Stained protein bands were The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: Rsd, regulator of sD; GST, glutathione S-transferase; accordance with 18 U.S.C. §1734 solely to indicate this fact. LB, Luria–Bertani. © 1998 by The National Academy of Sciences 0027-8424y98y954953-6$2.00y0 *To whom reprint requests should be addressed. e-mail: aishiham@ PNAS is available online at http:yywww.pnas.org. lab.nig.ac.jp. 4953 Downloaded by guest on September 24, 2021 4954 Biochemistry: Jishage and Ishihama Proc. Natl. Acad. Sci. USA 95 (1998) Table 1. Primers used for gene amplification Gene Primer Sequence rpoD (s70 gene) D1 59-CCCAAGCTTGAATTCATGGAGCAAAACCCGCAGTCAC-39 D4 59-CCCAAGCTTGTCGACTTAATCGTCCAGGAAGCTACG-39 rpoN (s54 gene) N1 59-GGAATTCCATATGAAGCAAGGTTTGCAACTCAGG-39 N2 59-CATGCATGCCTCGAGTCAAACGAGTTGTTTACGCTG-39 rpoS (s38 gene) S5 59-GCGAATTCCATATGTTCCGTCAAGGGATCACG-39 S6 59-GCGGATCCCTCGAGTTACTCGCGGAACAGCGCTTC-39 rpoF (s28 gene) F1 59-CGAGCTCGGATCCCCATGAATTCACTCTATACCGCT-39 F2 59-GGGGTACCCTCGAGTTATAACTTACCCAGTTTAGT-39 flgM (FlgM gene) M1 59-GCGAATTCCATATGAGTATTGATCGCACTTCGCC-39 M2 59-GCGGATCCCTCGAGGTTACTCTGCAAGTCTTGCTG-39 rsd (Rsd gene) RsdU 59-GCGAATTCCATATGCTTAACCAGCTCGATAACC-39 RsdR 59-AACTGCAGCTCGAGAGCAGGATGTTTGACGCGGGC-39 directly subjected to automated microsequencing (Applied Bio- ketone-treated trypsin (Sigma), and 1 nmol of sample protein. systems 491 protein sequencer). Purification and analysis of The reaction was carried out for 20 min at 25°C and then anti-s-associated proteins were carried out by the same proce- terminated by adding 5 ml of 100 mM phenylmethylsulfonyl dure by using pGEXM and pGEXRD. fluoride. Reaction products were analyzed by SDSyPAGE on Preparation of s Subunits and Rsd. Overexpression and 5–15% gradient gels. purification of s70, sN, sS, sH, sF, and sE were performed as In Vitro Single-Round Transcription Assay. RNA polymerase described previously (19–21). To overproduce the His6-tagged or core enzyme was purified from E. coli W3350 by passing the GST-fused Rsd proteins, an E. coli BL21(lDE3) transformant purified RNA polymerase at least three times through phospho- containing pET-Rsd or a strain DH5 transformant containing cellulose columns (22). Holoenzymes were reconstituted by mix- pGEX-Rsd was grown at 37°C in LB medium containing ampi- ing the core enzyme and 3-fold molar excesses of s subunits. cillin, and when the culture reached 30 Klett units (540-nm filter), Single-round transcription by the holoenzyme was carried out isopropyl b-D-thiogalactopyranoside was added at 1 mM. After under the standard conditions described previously (23) except 1 h, cells were harvested and stored at 280°C until use. Cell that the concentrations of templates and RNA polymerase were lysates were prepared essentially as described previously (19). For varied as indicated in each experiment. In brief, mixtures of the purification of His6-tagged Rsd protein, the soluble fraction template and RNA polymerase were preincubated at 37°C for 30 was applied onto a Ni21-nitrilotriacetic acid column (Qiagen) min to allow open complex formation in 35 ml of the standard equilibrated with a binding buffer (20 mM TriszHCl, pH 8.0y0.5 transcription mixture containing 50 mM NaCl. RNA synthesis M NaCl). The column was washed with 103 bed volumes of the was initiated by adding a 15-ml mixture of substrate and heparin binding buffer containing 5 mM imidazole, followed by elution in the standard transcription buffer and continued for 5 min at with a step gradient of imidazole from 10 to 200 mM.
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