Nucleic Acids Research, 2000, Vol. 28, No. 18 3497-3503
© 2000 Oxford University Press
Competition among seven Escherichia coli 
subunits: relative binding affinities to the core RNA polymerase
1National Institute of Genetics, Department of Molecular Genetics, Mishima, Shizuoka 411-8540, Japan and 2Kagoshima University, Faculty of Fisheries, Kagoshima 890-0056, Japan
Received June 2, 2000; Revised and Accepted July 18, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Seven different species of the RNA polymerase
subunit exist in Escherichia coli, each binding to a single species of the core enzyme and thereby directing transcription of a specific set of genes. To test the competition model in the global regulation of gene transcription, all seven E.coli subunits have been purified and compared for their binding affinities to the same core RNA polymerase (E). In the presence of a fixed amount of 70, the principal for growth-related genes, the level of E70 holoenzyme formation increased linearly with the increase in core enzyme level, giving an apparent Kd for the core enzyme of 0.26 nM. Mixed reconstitution experiments in the presence of a fixed amount of core enzyme and increasing amounts of an equimolar mixture of all seven subunits indicated that 70 is strongest in terms of core enzyme binding, followed by N, F, E/FecI, H and S in decreasing order. The orders of core binding affinity between 70 and N and between 70 and H were confirmed by measuring the replacement of one core-associated by another subunit. Taken together with the intracellular levels, we tried to estimate the number of each holoenzyme form in growing E.coli cells. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The bacterial RNA polymerase is composed of the core enzyme (subunit composition
2ßß') with RNA polymerization catalytic activity and one of multiple species of the subunit for promoter recognition (1–6). In Escherichia coli, seven different species of the subunit, 70, N (also called 54), S (38), H (32), F (28), E (24) and FecI, are known to exist, each directing transcription of a specific set of genes. Most of the growth-related and housekeeping genes expressed in the exponential phase of cell growth are transcribed by the holoenzyme containing 70 (the rpoD gene product), while the holoenzyme ES is essential for transcription of some stationary phase-specific genes (7,8). The stress response genes are transcribed by RNA polymerase holoenzymes containing the alternative minor subunits. The holoenzyme EN transcribes genes which are regulated by the availability of nitrogen (9) and some stress response genes (10); the holoenzyme EH transcribes the genes for heat shock proteins (4,10); EF is needed for expression of the flagella and chemotaxis genes (11); the holoenzyme EE is responsible for transcription of the genes for extracytoplasmic functions as well as the heat shock response (12–14); the fecI gene product, which was originally identified as a regulatory gene for the ferric citrate transport system (15), is now known as a new member of the extracytoplasmic function (ECF) subfamily of factors on the basis of protein sequence (hereafter referred to as FecI) and is involved in transcriptional regulation of the genes for extracytoplasmic functions (16–18). We have purified all seven species of the E.coli subunit and analyzed their recognition specificity for various E.coli promoters (18–20). Using specific antibodies raised against the purified proteins, we also measured the intracellular concentrations of all seven subunits for both exponential and stationary phase cultures of E.coli W3110 (A) (20–22).
The global pattern of gene transcription is believed to be determined through competition between available subunits (23–25) and, if this is the case, replacement of one core enzyme-associated subunit by another should be the major determinant in switching of the global transcription pattern (reviewed in 26,27). In fact, the change in global transcription pattern during the growth phase transition from exponential to stationary phase (21,22) or upon sudden exposure to heat shock (28,29) is accompanied by a change in intracellular levels of the subunits. At present, however, it remains unsolved whether changes in concentrations alone can explain the change in transcription pattern. To gain an insight into the mechanism of switching we have performed a qualitative comparison of the binding affinities of all seven E.coli subunits for the same core enzyme. On the basis of this first systematic comparison of the binding affinity of each subunit for the core enzyme, together with determination of the intracellular concentrations of each subunit (20–22), an attempt was made to estimate the total number of each holoenzyme form in E.coli. On the basis of these observations, the competition model is evaluated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Overexpression and purification of
subunitsOverexpression of
70, N, S, F, H, E and FecI was performed using the expression plasmids pGEMD (30), pKES259 (31), pETF (18), pETSF (19), pET21H (S.Kusano and A.Ishihama, unpublished results), pRPOE (32) and pETFecI (20), respectively. Escherichia coli strain BL21(DE3) was transformed with the respective expression plasmid and grown in Luria–Bertani (LB) medium containing ampicillin (0.2 mg/ml). After induction with isopropyl ß-D-thiogalactopyranoside (IPTG), cells were harvested, washed with 10 mM Tris–HCl, pH 8.0, 1 mM EDTA and 150 mM NaCl and stored at –80°C until use. Cells were resuspended in lysis buffer (50 mM Tris–HCl, pH 8.0, 1 mM EDTA and 0.1 mM NaCl) and treated with phenylmethylsulphonyl floride (PMSF), lysozyme and sodium deoxycholate. The lysate was homogenized by gentle sonication and insoluble materials were recovered by centrifugation. All seven proteins were extracted from the inclusion bodies with elution buffer (50 mM Tris–HCl, pH 8.0, 0.1 mM EDTA, 0.1 mM DTT and 5% glycerol) containing 0.5% Triton X-100 or 0.5 M KCl. Purification of the solubilized subunits was performed without using protein denaturants essentially as described previously (30).
The protein concentration was determined with the Bradford protein assay kit (Bio-Rad) using bovine serum albumin as a standard. The relative content of each subunit in purified preparations was estimated by measuring the staining intensity with Coomassie brilliant blue (CBB) of the band after separation of contaminating proteins by SDS–PAGE. The purified subunits were stored frozen at –80°C in storage buffer [50 mM Tris–HCl (pH 7.6 at 4°C), 10 mM magnesium acetate, 0.1 mM EDTA, 1 mM DTT, 0.2 M KCl and 50% (v/v) glycerol] until use.
RNA polymerase core enzyme
RNA polymerase was purified from E.coli W3350 and the core enzyme was purified by passing the purified RNA polymerase three times through a phosphocellulose column. Repetition of the phosphocellulose column chromatography at least three times was needed to completely remove minor subunits from the core enzyme (33,34). The level of remaining subunits was double checked by testing for in vitro transcription activity directed by specific promoters and by immunostaining with antibodies raised against each subunit. The purified core enzyme was stored frozen at –80°C in storage buffer [50 mM Tris–HCl (pH 7.6 at 4°C), 10 mM magnesium acetate, 0.1 mM EDTA, 1 mM DTT, 0.2 M KCl and 50% (v/v) glycerol] until use.
Reconstitution of holoenzymes
The core enzyme and a single species or combinations of various subunits were mixed in 50 µl of reconstitution buffer [10 mM Tris–HCl (pH 7.6 at 4°C), 0.1 mM DTT, EDTA, 0.1 mM, 200 mM NaCl and 5% glycerol] and incubated for 10 min at 30°C. Aliquots of 40 µl were subjected to gel filtration through a Superdex 200 column PC3.2/300 (bed volume 0.9 ml) with a SMART system (Pharmacia, Sweden). Elution with reconstitution buffer was performed at a flow rate of 40 µl/min at 20°C. Both holoenzyme and core enzyme were recovered in the void volume (elution time 22–23 min). Aliquots of each elution fraction were analyzed by SDS–PAGE. Gels were stained with a SYPRO Orange staining kit (Molecular Probes, USA). The protein concentration was determined by scanning the gels with a FluorImager (Vistra, USA). The concentrations of RNA polymerase proteins determined with the SYPRO Orange staining kit paralleled those stained with CBB, although the sensitivity of detection is much higher for SYPRO staining. The level of holoenzyme formation was determined by measuring the ratio between the (the representative subunit of the core enzyme) and subunits in the RNA polymerase peak (holoenzyme plus core enzyme).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dissociation constant of E
70 holoenzyme formationThe RNA polymerase holoenzyme can be formed in vitro by mixing purified core enzyme and
subunit. For comparison of the binding affinities of different subunits for the core enzyme, we purified the -free core enzyme by passing the purified RNA polymerase three times through phosphocellulose columns (33,34). All seven subunits were purified from E.coli cells, which were induced for transient expression of each subunit, without using protein denaturants as in the case of 70 purification (30). The formation of holoenzyme was measured by mixing the purified core enzyme and the purified subunit(s) under various conditions. The amount of core enzyme-bound subunit(s) was determined by measuring the molar ratio of to subunits in the RNA polymerase peak (holoenzyme plus core enzyme) after gel filtration column chromatography.
First we determined the equilibrium dissociation constant of E70 holoenzyme formation (E represents the core enzyme). A constant amount (0.02 pmol/50 µl or 0.4 nM) of the 70 subunit and increasing amounts of the core enzyme were mixed at 0°C and, after 10 min incubation at 30°C to establish equilibrium, the reconstituted E70 holoenzyme was quickly separated, within 22–23 min, from unbound 70 by gel filtration through a Superdex 200 column using a SMART system (Pharmacia, Sweden). Figure 1A shows the level of core-bound 70 as determined by measuring the molar ratio between the 70 and subunits in the RNA polymerase peak. In the presence of excess amounts of the core enzyme virtually all the purified 70 subunits formed the E70 holoenzyme (see below), indicating that the 70 preparation used was fully active in binding to the core enzyme. Since dissociation of preformed E70 is virtually negligible within this time range, the apparent dissociation constant (Kd) of 70 from the core enzyme was estimated to be 
0.26 nM (Fig. 1A). To confirm the high level activity of 70 in core enzyme binding, we performed holoenzyme reconstitution with increasing amounts of the 70 subunit in the presence of an excess (20 pmol in 50 µl or 400 nM) of the core enzyme. The input 70 was quantitatively bound to the core enzyme up to 10 pmol and almost 75% of the input core enzyme was converted to the holoenzyme (E70) by adding 20 pmol of 70 (an input molar ratio of 1) (Fig. 1B). On adding a 10-fold molar excess of the 70 subunit all the input core enzyme was converted to the holoenzyme form. The amount of 70 required for saturation of the core enzyme was slightly more than that expected from the Kd value, presumably because some of 70 and/or core enzyme formed aggregates under such high protein concentrations.
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The finding that almost all the input
70 subunit was reconstituted to holoenzyme by adding excess amounts of the core enzyme (see Fig. 1A) indicates that virtually all the molecules in the 70 preparation used are active, at least in binding to the core enzyme. The holoenzyme formed from the 70 molecules was, however, not fully active in transcription as measured using a single round in vitro transcription assay directed by the lacUV5 promoter, implying that some of the inactive 70 subunit with respect to overall transcription is still active in binding to the core enzyme. Thus the dissociation constant (Kd) of E70 formation should be <0.26 nM.
Competition in core enzyme binding among seven subunits
For comparison of the core enzyme binding affinities among seven E.coli subunits we purified all other six subunits from overexpressed E.coli cells to apparent homogeneity (Fig. 2A). To recover all seven subunits in active forms, care was taken to purify the subunits by the same procedure from overexpressed cell extracts, i.e. extraction of the proteins from the pellet fraction of overexpressed cell extracts using a solubilization buffer containing a non-ionic detergent, without using ionic detergents. The concentration of each subunit was determined after correction for purity (90–98%), as estimated by SDS–PAGE followed by CBB staining. Since all these purified subunits were completely converted into the respective holoenzymes by adding excess amounts of the core enzyme (data not shown), the preparations used were fully active, at least in binding to the core enzyme.
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Using the purified
subunits, we first analyzed the saturation curve of each subunit to convert a fixed amount (20 pmol) of the core enzyme to the respective holoenzyme. At an input molar ratio of 1 more than 80% of the input core enzyme was converted to the holoenzyme for five subunits, 70, N, F, E and FecI, and 60–65% of the input core was converted to the holoenzyme for S and H (data not shown). For saturation of the input core enzyme, higher protein concentrations were required for both S and H. The input core enzyme was saturated with addition of a 2-fold molar excess of even for S and H, with weak binding affinities. A similar order of activity was obtained when the level of functional holoenzyme was determined by measuring the saturation curve using in vitro transcription assays directed by specific promoters for each subunit (data not shown). In the presence of single additions, however, it was difficult to determine the slight difference in core binding affinity among the seven subunits.
In order to measure the relative affinity of core enzyme binding among seven subunits we next carried out a mixed reconstitution experiment in the presence of all seven subunits in the same reaction mixture. To 20 pmol of the core enzyme were added increasing amounts of an equimolar mixture of all seven subunits and the subunits were fractionated into core enzyme-bound and unbound fractions by gel filtration column chromatography (Fig. 2B). The core enzyme-associated subunits were determined after measuring the molar ratios between the subunit and each subunit in the holoenzyme peak. Almost 68% of the core enzyme was converted into the holoenzymes at an input molar ratio of 0.25 for each subunit (or 1.75 for the combined subunits) (Fig. 3A) and >90% of the input core enzyme was bound with one of the seven subunits at an input molar ratio of 1.0 for each subunit (or 7.0 for the combined subunits) (Fig. 3B). The input core enzyme was saturated with one of the subunits at an input molar ratio of 2.0 for each subunit (or 14 for the combined subunits) (Fig. 3C). The core enzyme-associated fraction was, however, significantly different among the seven subunits. At low concentration (or 60–70% saturation level) N showed an affinity as high as that of the 70 subunit and the combined EN and E70 level was as high as 60% of the total holoenzymes. The holoenzyme level was almost the same among EF, EFecI and EE, each constituting 14, 10 and 8%, respectively, while the amount of EH was <1% of total holoenzymes. The level of ES formation was lowest under the reconstitution conditions employed.
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The core enzyme-bound fractions increased in parallel for all seven
subunits concomitantly with the increase in input subunits to a fixed amount of the core enzyme (Fig. 3B and C). At saturation the order of core enzyme-associated subunits was 70 > N > F > H/FecI > E > S. Under saturation conditions the fraction of E70 holoenzyme was 39% of the total holoenzymes. We then conclude that under competitive binding conditions in the presence of all seven subunits the affinity of 70 is strongest and that of S is weakest. Judging from the affinity difference (>16-fold) in core enzyme binding between 70 and S and the Kd value of 0.26 nM for 70 (see above), we estimate that the apparent Kd value of S for the core enzyme is 4.3 nM (Table 1).
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Switching of one core enzyme-bound
for another subunitFrom the binding affinity of each
subunit determined as above we could predict the efficiency of replacement of one core enzyme-bound subunit by another subunit. Here we tested two extreme combinations: (i) 70 and H which has a 10-fold lower binding affinity for the core enzyme than 70; and (ii) 70 and N which has a core enzyme binding affinity as high as 70. An equimolar mixture of core enzyme (20 pmol) and one subunit (20 pmol) was incubated to equilibrium and then an equimolar amount (20 pmol) of the second subunit was added. At various times after addition of the second the core enzyme-associated subunits were measured. Results of 70/H competition experiments indicated that: there was little replacement of the core enzyme-associated 70 by the added H even after 600 min incubation (Fig. 4A); core-bound H was rapidly replaced by 70 within 10 min (Fig. 4B). On the other hand, in the competition experiment between 70 and N 30% of the core enzyme-associated 70 was replaced by the added N (Fig. 4C) while at least half of the core-bound N remained associated even after prolonged incubation for 600 min following addition of 70 (Fig. 4D). All these results are in good agreement with the differences in core enzyme binding affinity between 70 and H and between 70 and N (Fig. 3; see also Table 1). Moreover, the slight difference in the rate of replacement between the two combinations, E70/EN (30% in Fig. 4C) and EN/E70 (50% in Fig. 4D), reflects the slight difference in the core enzyme binding affinity between 70 and N (see Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Difference in the core enzyme binding affinity among seven E.coli
subunitsThe comparison of core binding affinity among seven
subunits from E.coli indicates that the 70 subunit for transcription of growth-related and housekeeping genes has the highest affinity for the core enzyme. The affinity difference was 16-fold between 70 (0.26 nM) and S, with the weakest binding activity (4.28 nM) (see Table 1). This estimation relied on the observation that the content of functional molecules with respect to core enzyme binding was the same between the seven subunit preparations. Some of the molecules with core enzyme binding activity are, however, inactive in carrying out the complete cycle of transcription. In the case of the 70 subunit, for instance, the content of transcriptionally competent molecules was 50% as measured by the synthesis level of template sized lacUV5 RNA in a single round transcription assay. This value of fully functional 70 subunit represents the minimum, because: (i) a certain fraction of initiation complexes with E70 become abortive prior to promoter escape (the level of abortive initiation depends on the nature of the promoter) (see for instance 35); (ii) some transcriptionally inactive E70, as determined using the lacUV5 promoter-directed transcription assay, could be active with promoters other than lacUV5. The precise estimation of functional molecules is more difficult for other subunits because available promoters recognized by these subunits are limited and because we do not know the ratio of productive versus abortive initiation of in vitro transcription catalyzed by each holoenzyme. However, we took maximum care to obtain the other six subunits in as active a form as the 70 subunit, by employing the same procedures for protein expression and purification as used for the 70 subunit. As a result, all the preparations used were fully active in binding to the core enzyme.
The purity of the seven subunits ranged from 90 to 98%, as detected by SYPRO Orange staining, which is as sensitive as silver staining. Contamination of preparations by the anti- factors Rsd (36), FlgM (37) and Rse (38,39) would result in a decrease in the concentration of functional molecules or interference with –core enzyme interactions. However, an immunoblot analysis of the preparations used in this study did not give any signal against anti-Rsd and anti-FlgM antibodies (data not shown).
The Kd value (0.26 nM) for 70 herein determined is slightly lower than a previous estimation (0.5–1.0 nM) using HPLC gel filtration and fluorescence techniques (40). The difference might be due to a difference in content of the functional 70 subunit and/or core enzyme or the assay conditions. Joo et al. (41) estimated a Kd value of 1 nM for H from a saturation of in vitro transcription experiment, but of 100 nM by glycerol gradient centrifugation. The binding constants obtained by both gel filtration and glycerol gradient centrifugation should be lower than that obtained in transcription assays, because of the dilution effect during separation of core enzyme-bound from unbound free . Thus, further technical improvements are needed for accurate determination of the binding affinity of each subunit for the core enzyme, but the present study provides the relative order of core enzyme binding affinity among seven E.coli subunits. The present study clearly shows that the affinity of the N subunit for the core enzyme is as high as those of other subunits. Thus the ATP-dependent reaction in formation of the EN–promoter open complex may be at a step after binding of the EN holoenzyme to the promoter.
Structural basis of the difference in core enzyme binding affinity between subunits
The 70 family of proteins have a common structural organization, except for 54, consisting of four conserved domains (4,5). The role of each domain in promoter recognition has been extensively studied employing genetic, chemical and physical approaches (for a review see 4). On the other hand, our knowledge of the role of structural domains in protein–protein interactions with the core enzyme is limited. We have identified the 70, N and S subunit contact surfaces on the core enzyme subunits after mapping contact-dependent cleavage sites with iron(S)-1-(p-bromoacetamidobenzyl)ethylenediamine tetraacetate (Fe-BABE) (42) tethered at various positions on the subunits (43–45). The results indicated that multiple sites along these polypeptides are involved in contact with the ß and ß' subunits of the core enzyme, most of which are located in the conserved domains among the three subunits. Mutant studies also indicated that the 70 subunit contains multiple contact interfaces with the core enzyme (46) and, moreover, the same regions, and even equivalent amino acid residues, in both 70 and H are involved in core enzyme binding. If the major interfaces for protein–protein contacts with the core enzyme are the same within the family of proteins, the differences in core enzyme binding affinity observed in this study may be attributed to differences in the number of contact surfaces or contact amino acid residues or in the affinity of minor interfaces characteristic of each subunit. The contact-dependent protein cleavage experiments with FeBABE indeed indicated the presence of small numbers of unique core subunit contact sites characteristic of each subunit (43–45).
Intracellular levels of various holoenzyme forms of E.coli RNA polymerase
The intracellular concentration in E.coli W3110 (A) is highest for the 70 subunit among the seven E.coli subunits in both the exponential and stationary phases and under various stress conditions (20–22). From the dissociation constants of core enzyme binding and the intracellular concentrations we are now able to estimate the intracellular concentration of each holoenzyme (see Table 1). The concentration of RNA polymerase in E.coli W3350 is 2000 molecules/cell in the exponential phase, of which about one-third (700 molecules) stays in the cytosol (3,6). Since the total number of combined subunits (1200 molecules/cell) is more than that of RNA polymerase not engaged in the transcription cycle (700 molecules/cell), the majority of the RNA polymerase in the cytosol must be in the holoenzyme form associated with one of the subunits. For instance, in exponential phase E.coli W3110 cells the numbers of each holoenzyme can be calculated to be 545 molecules for E70, 100 molecules for EF and 55 molecules for EN.
However, some of the 70, F and N subunits are considered to exist as complexes with anti- factors (24,36). Thus, the actual concentrations of the E70, EF and EE holoenzymes in E.coli should be lower than the levels estimated above. For instance, a considerable fraction of the F subunit stays as a complex with FlgM, the anti-F factor, under the same culture conditions as employed in this study (36,37). E activity is regulated by RseA (regulator of E or anti-E factor), which is associated with the inner membrane and inhibits the activity of E by directly interacting with E (38,39). Recently we identified Rsd (regulator for D), the putative anti-70 subunit, which is produced in E.coli during the transition from the exponential to the stationary phase (25,36). The control of functional forms of the subunits by anti- factors or switching factors may contribute, at least to a certain extent, in regulation of the relative levels of various forms of the holoenzyme (6,27,47).
Factors affecting the core enzyme binding affinity of subunits
competition has been shown to take place between the E.coli 70 subunit and the phage T4 subunit (48–50). In uninfected cells, however, the model has been accepted without any quantitative measurements of the core enzyme binding affinity for each subunit and of the intracellular concentrations of the seven subunits. The observation that overexpression of one subunit affects transcription of genes under the control of other subunits (24,25) supports the competition model. Judging from the intracellular concentrations of all seven subunits (20–22) and the binding affinity of each subunit for the core enzyme (this paper), it became clear that the subunits compete for a limited number of core enzyme molecules. However, both the intracellular level and the binding affinity for the core RNA polymerase are highest for 70, the major subunit for transcription of growth-related genes, at least under the experimental conditions employed, indicating that competition alone cannot explain efficient replacement of the 70 subunit by the alternative subunits.
Previously we proposed that, to explain the growth-coupled change in transcription pattern, some specific intracellular conditions or additional factors are involved in efficient replacement of the core enzyme-associated subunit (26,27). For instance, a growth-dependent change in cytosol composition such as an increase in glutamate (51), trehalose (52) or polyphosphate (53) can promote preferential utilization of S over 70. Regulatory nucleotides such as cAMP, ppGpp and AppppA may also affect transcription by different holoenzymes in different ways (54). Along these lines, the binding affinity of each subunit to the core RNA polymerase should be examined under various conditions mimicking the intracellular conditions for each subunit function. The major factor(s) affecting replacement awaits further studies.
In addition to switching control at the step of core enzyme binding, the recruitment frequency of various holoenzymes into the transcription cycle is also subject to control. For instance, a growth-coupled change in DNA superhelicity affects the utilization of E70 and E38 in different ways (34,55). The protein composition of the nucleoid also changes markedly depending on cell growth conditions (56,57). Most of the nucleoid proteins are known to be global regulators of gene transcription, influencing transcription by various holoenzymes in different manners.
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ACKNOWLEDGEMENTS |
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We thank S. Kusano, T.S. Kundu, M. Jishage and T. Nomura for preparation of
S, H, F and E, respectively. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan and CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +81 559 81 6741; Fax: +81 559 81 6746; Email: aishiham@lab.nig.ac.jp
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