Assay of Prokaryotic Enhancer Activity over a Distance In Vitro

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[29] Assay of Prokaryotic Enhancer Activity over a Distance In Vitro By Vladimir Bondarenko, Ye V. Liu, Alexander J. Ninfa, and Vasily M. Studitsky Regulation of expression of eukaryotic genes depends almost entirely on enhancers—30- to 200-bp DNA sequences usually composed of several binding sites for an activator protein(s). The landmark of enhancers is their ability to activate target genes over a large distance (up to 60 kb).1 Analysis of eukaryotic transcriptional enhancers is complicated by the fact that current RNA polymerase (RNAP) II-dependent experimental systems in vitro are very inefficient (less than 1% of templates are transcribed2). In contrast, transcriptional enhancers of Escherichia coli are extremely efficient and, under the appropriate conditions, can activate transcription of the majority of DNA templates in vitro.3,4 Prokaryotic and eukaryotic enhancers share several key properties: activation over a large distance, tight coupling of DNA melting with ATP hydrolysis, high stability of the initiation complexes, and absolute dependence of transcription on the presence of an activator are crucial features of eukaryotic transcription machinery shared only by enhancer-dependent promoters in E. coli (see Buck et al.5 for review). These properties of prokaryotic enhancers make them a valuable system for the investigation of enhancer function, providing data complementary to in vivo studies of their eukaryotic counterparts. The mechanism of action of bacterial transcriptional enhancers has been studied intensely using the glnAp2 promoter of E. coli as a model system. Activity of this promoter is entirely dependent on the activator,
,54 and, under the appropriate conditions in vitro, is dependent on the enhancer.3,4,6 The enhancer is bound by the phosphorylated form of the activator protein NtrC, which is phosphorylated by the NtrB protein kinase, as well as by acetyl phosphate.7,8 When phosphorylated, NtrC 1

E. M. Blackwood and J. T. Kadonaga, Science 281, 61 (1998). J. A. Knezetic, G. A. Jacob, and D. S. Luse, Mol. Cell. Biol. 8, 3114 (1988). 3 A. J. Ninfa, L. J. Reitzer, and B. Magasanik, Cell 50, 1039 (1987). 4 D. L. Popham, D. Szeto, J. Keener, and S. Kustu, Science 243, 629 (1989). 5 M. Buck, M. T. Gallegos, D. J. Studholme, Y. Guo, and J. D. Gralla, J. Bacteriol. 182, 4129 (2000). 6 S. Sasse-Dwight and J. D. Gralla, Proc. Natl. Acad. Sci. USA 85, 8934 (1988). 7 A. J. Ninfa and B. Magasanik, Proc. Natl. Acad. Sci. USA 83, 5909 (1986). 8 J. Keener and S. Kustu, Proc. Natl. Acad. Sci. USA 85, 4976 (1988). 2

METHODS IN ENZYMOLOGY, VOL. 370

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forms homooligomers, interacts with the E
54 holoenzyme, and stimulates conversion from the closed (RPc) to the open (RPo) complex.4,6,9–12 During the enhancer–promoter interaction, intervening DNA is looped out transiently,13,14 placing enhancer and promoter in close proximity to each other.15 The progress of studies of the mechanism of prokaryotic enhancer action was somewhat limited by the lack of an experimental system supporting multiple-round transcription. In particular, some models for the action of eukaryotic enhancers (such as the ‘‘hit-and-run’’ and ‘‘stable DNA loop’’ models16) propose existence of a ‘‘molecular memory’’ facilitating enhancer action during multiple-round transcription (see Blackwood and Kadonaga1 for review). This class of the models can only be analyzed properly using a multiple-round transcription assay. We have established an experimental system supporting the action of prokaryotic enhancers over a large distance (more than 2.5 kb) and allowing multiple rounds of transcription per template in vitro.17–19 Using this system, we have demonstrated that DNA supercoiling greatly facilitates enhancer–promoter communication over a large distance and eliminated other previously proposed ‘‘memory’’ models for enhancer action over a distance.17,18 We have also shown that low-affinity NtrC-binding sites between the enhancer and the promoter act as a ‘‘governor’’ to reduce the rate of transcription initiation at very high activator concentrations.19 This article describes key experimental techniques used for the analysis of the mechanism of enhancer action. Materials

MicroSpin G-50 column, Sephacryl S-400, and heparin–Sepharose (Pharmacia); hydroxyapatite, Bio-Rex 70, and silver stain kit (BioRad); plasmid purification mega kit (Qiagen) 9

M. Buck and W. Cannon, Mol. Microbiol. 6, 1625 (1992). S. C. Porter, A. K. North, A. B. Wedel, and S. Kustu, Genes Dev. 7, 2258 (1993). 11 A. Wedel and S. Kustu, Genes Dev. 9, 2042 (1995). 12 C. Wyman, I. Rombel, A. K. North, C. Bustamante, and S. Kustu, Science 275, 1658 (1997). 13 W. Su, S. Porter, S. Kustu, and H. Echols, Proc. Natl. Acad. Sci. USA 87, 5504 (1990). 14 K. Rippe, M. Guthold, P. H. von Hippel, and C. Bustamante, J. Mol. Biol. 270, 125 (1997). 15 A. Wedel, D. S. Weiss, D. Popham, P. Droge, and S. Kustu, Science 248, 486 (1990). 16 V. M. Studitsky, FEBS Lett. 280, 5 (1991). 17 Y. Liu, V. Bondarenko, A. Ninfa, and V. M. Studitsky, Proc. Natl. Acad. Sci. USA 98, 14883 (2001). 18 V. Bondarenko, Y. Liu, A. Ninfa, and V. M. Studitsky, Nucleic Acids Res. 30, 636 (2002). 19 M. R. Atkinson, N. Pattaramanon, and A. J. Ninfa, Mol. Microbiol. 46, 1247 (2002). 10

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Reagents

Ethidium bromide; butyl alcohol; ethanol; NaCl; 2-mercaptoethanol; urea; heparin; chloroform; KMnO4 (Sigma); RNase inhibitor (Roche); ATP, UTP, CTP, GTP, dATP, dUTP, dCTP, dGTP (Pharmacia); [-32P]UTP, 3000 Ci/mmol, and [-32P]ATP, 6000 Ci/ mmol (NEN); Tris-saturated phenol (Invitrogen); sheared DNA (Intergen) Buffers

Transcription buffer (TB): 50 mM Tris–OAC pH 8.0, 100 mM KOAc, 8 mM Mg (OAc)2, 27 mM NH4OAc, 0.7% PEG (8000), and 0.2 mM dithiothreitol (DTT) 2
transcription stop buffer (2XTSB): 200 g/ml sheared DNA, 40 mM EDTA TMD buffer: 50 mM Tris–Cl, pH 7.2, 10 mM MgCl2, 0.2 mM DTT Quench buffer (QB): 4 M NH4OAc, 20 mM EDTA Protein purification buffer (PPB): 50 mM Tris, pH 8.0, 5% glycerol, 10 mM EDTA, 1 mM DTT

Enzymes

Escherichia coli enzymes: Core RNAP;
54; NtrC; NtrB (purified as described later); Klenow fragment (DNA polymerase I large fragment, Invitrogen); DNase I (Sigma); calf thymus DNA topoisomerase I (Invitrogen); T4 polynucleotide kinase (New England Biolabs) Purification of DNA, Proteins, and Protein Complexes

Plasmid templates are purified using routine techniques.17,18 NtrC and
are purified using previously published protocols.20,21 Methods for isolation of core RNAP and NtrB20,22 are modified considerably and are described in detail later. 54

20

T. P. Hunt and B. Magasanik, Proc. Natl. Acad. Sci. USA 82, 8453 (1985). L. J. Reitzer and B. Magasanik, Proc. Natl. Acad. Sci. USA 82, 1979 (1985). 22 A. J. Ninfa, S. Ueno-Nishio, T. P. Hunt, B. Robustell, and B. Magasanik, J. Bacteriol. 168, 1002 (1986). 21

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Purification of E. coli Core RNAP This is a modification of the protocol described by Burgess and Jendrisak.23 This method involves French press lysis, polymin precipitation, elution of the enzyme from the polymin precipitate with high salt, ammonium sulfate precipitation, and chromatography on heparin–Sepharose and BioRex 70. Core RNA polymerase is purified to electrophoretic homogeneity (more than 95% purity) with a yield of 32 mg of core enzyme from 100 g of  E. coli cells. All procedures are conducted at 4 . This method provides core polymerase that is suitable for transcription studies. For purer core polymerase, we used a BioGel 0.5 M gel filtration step between the heparin–Sepharose and Bio-Rex 70 steps, as described.23 As an alternative to using polymerase purified as described later, core RNAP from Epicentre Technologies may be used. However, we have observed that this preparation of core polymerase is contaminated with
54, such that it is not possible to conduct control experiments lacking
54. 1. Thaw 100 g of frozen E. coli cells (JM105 strain), homogenize in 200 ml ice-cold PPB buffer supplemented with 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and lyse the cells by passing twice through a French press at 12,000 psi. 2. Remove cell debris by centrifugation on a Sorvall rotor GS3 at 8000 rpm for 45 min. Carefully transfer the clear supernatant into a glass beaker and measure its volume. 3. Slowly add (with constant stirring) 10% polymin P, pH8.0, to a final concentration 0.4%. Continue stirring for 10 min. 4. Precipitate aggregated DNA–protein complexes by centrifugation at 8000 rpm for 15 min. Remove the supernatant, scrape the pellet into a prechilled low-speed Hamilton blender with 250 ml of PPB containing 0.5 M NaCl, and resuspend pellet with gentle stirring for 5–10 min. Remove supernatant and wash the pellet one more time with PPB buffer containing 0.5 M NaCl. 5. Remove supernatant and dissolve precipitate in 200 ml of PPB buffer containing 1 M NaCl using a blender as described earlier. Add ammonium sulfate powder to the 1 M NaCl eluate with stirring to 50% saturation (35 g 100 ml). When salt is dissolved completely, continue stirring for 20 min.

23

R. R. Burgess and J. J. Jendrisak, Biochemistry 14, 4634 (1975).

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6. Centrifuge the mixture at 8000 rpm for 15 min. Discard supernatant and dissolve drained pellet in 25 ml of PPB buffer containing 10 mM MgCl2 and 50 mM NaCl. Dialyze against 1 liter of the same buffer for 6 h. 7. Clear the dialyzed solution to remove formed precipitate at 15,000 rpm on a Sorvall SS-34 rotor for 10 min and load cleared supernatant on a 15-ml (8.5
1.5 cm) heparin–Sepharose column equilibrated with PPB buffer containing 10 mM MgCl2 and 50 mM NaCl buffer. Wash column with 60 ml of the same buffer and elute RNA polymerase with a 100-ml linear salt gradient from 0.05 to 0.5 M NaCl at a flow rate of 0.5 ml/ min collecting 2-ml fractions. A peak of RNA polymerase, identified by 10% SDS–PAGE (AA:Bis ratio 29:1), elutes at about 0.25 M NaCl. Pool fractions containing RNA polymerase and dialyze overnight against 2  liters of PPB buffer containing 0.15 M NaCl at 4 . 8. Load dialyzed fractions on a 125-ml (30
4 cm) Bio-Rex 70 column equilibrated with PPB buffer containing 0.15 M NaCl. Wash the column with 200 ml of the same buffer and elute with 400 ml of a linear salt gradient from 0.15 to 1.0 M NaCl at a flow rate of 1 ml/min collecting 4-ml fraction. Analyze eluate by 10% SDS–PAGE to identify fractions containing core RNA polymerase. The
70 subunit comes out in a flowthrough fraction, and the core RNA polymerase is eluted at about 0.4 M NaCl. 9. Combine fractions containing core enzyme and dialyze against 10 mM Tris, pH 8.0, 50% glycerol, 0.1 mM EDTA, 0.1 mM DTT, 100 mM  NaCl overnight. Freeze fractions in liquid nitrogen and store at 70 . Expression and Purification of Recombinant E.coli NtrB The NtrB protein is isolated from E.coli strain RB9131R carrying plasmid pLOP, which contains the glnL (encoding the NtrB protein) gene under control of the PL promoter and temperature-sensitive  repressor. Ammonium sulfate fractionation is followed by DE52 and hydroxyapatitechromatography. The expression of NtrB is induced by raising the temperature during growth of the bacterial culture (in the middle of log phase)   from 30 to 44 to release the inhibitory effect of the  repressor on transcription of the glnL gene. The recombinant NtrB protein is isolated with >95% purity, resulting in a yield of about 12 mg of the enzyme from 8 g of  induced cells. All procedures are conducted at 4 . The NtrB purified as described later is free of detectable nuclease activity or other confounding activities and is suitable for transcription studies. The strongest step in the fractionation procedure is ammonium sulfate

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fractionation, after which NtrB is about 70–80% pure. As an alternative to the hydroxyapatite step, gel filtration on Sephadex G-100 may be used as the final step. For optimal purity of NtrB, chromatography on agaroseethane is used as an initial chromatography step22; however, this is not required for transcription studies. 1. Grow RB9131R/pLOP in 1 liter of LB medium containing   100 g/ml ampicillin at 30 to OD600  1.0, raise the temperature to 44 , and continue incubation for additional 4 h to induce expression of NtrB. 2. Harvest cells by centrifugation on a Sorvall GS-3 rotor at 8000 rpm for 15 min. 3. Resuspend cells (8 g total) in 25 ml of PPB buffer containing 50 mM Tris–HCl, pH 8.0, 5% glycerol, 1 mM EDTA, and 0.1 mM DTT. Lyse cells by passing twice through a French press at 12,000 psi and remove cell debris by centrifugation at 12,000 rpm (20,000 g) for 30 min on a Sorvall SS-34. 4. Slowly add ammonium sulfate powder to the cleared lysate, with constant stirring, until the first precipitate is formed (when the concentration of ammonium sulfate reaches 2 %) and continue stirring for an  hour or overnight at 4 . Spin down precipitate at 12,000 rpm for 30 min. 5. Discard the supernatant and resuspend drained pellet in 15 ml of PPB buffer. Dialyze mixture overnight against 0.5 liter of the same buffer and centrifuge dialyzes solution at 12,000 rpm for 20 min to remove precipitate formed during dialysis. 6. Load 15 ml of the cleared supernatant containing NtrB on a 35-ml (12
2 cm) DE52 column at a flow rate of 0.5 ml/min equilibrated with PPB buffer containing 25 mM NaCl, wash column with 70 ml of the same buffer, and elute with a 200-ml linear salt gradient from 0.025 to 1.0 M NaCl at a flow rate of 0.5 ml/min. NtrB is eluted at 0.25 M NaCl. 7. Combine fractions containing NtrB (identified by SDS–PAGE) and add an equal volume of 100% saturated ammonium sulfate with constant stirring. Incubate in ice for an hour and spin down precipitate at 20,000 g for 30 min. Discard supernatant and resuspend drained pellet in 12 ml of PPB buffer. Spin mixture to remove insoluble material at 12,000 rpm for 30 min and dialyze supernatant overnight against 1.0 liter  of 10 mM Na-phosphate buffer, pH 7.0 at 4 . 8. Load the dialyzed solution on a 12-ml (4
2 cm) hydroxyapatite column at a flow rate of 0.3 ml/min equilibrated with 10 mM Naphosphate, pH 7.0, and elute with 100 ml of linear gradient of Na-phosphate, pH 7.0, from 10 to 500 mM collecting 2-ml fractions.

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Fig. 1. Analysis of purified proteins used for reconstitution of enhancer-dependent transcription in vitro. Proteins were separated in 10% PAGE and stained with Coomassie. Mobilities of protein subunits are indicated on the right. M, protein molecular mass markers.

9. Pool fractions containing NtrB (identified by 10% SDS–PAGE) and dialyze overnight against buffer containing 50 mM Tris–HCl, pH 7.5, 50% glycerol, and 0.1 mM EDTA. Freeze in liquid nitrogen and store at  70 (up to several years without significant loss of activity). 10. Analyze purified proteins in 10 % SDS–PAGE (Fig. 1). Single- and Multiple-Round Assays for Enhancer-Dependent Transcription

After purification of the proteins required for specific enhancerdependent transcription, the whole system has to be characterized extensively using a single-round transcriptional assay (Fig. 2A). Single- and multiple-round transcription assays are also useful for analysis of different aspects of enhancer action3,4,17,18 (see later); an example is shown in Fig. 2B. The multiple-round transcription assay is particularly useful for analysis of kinetic aspects of enhancer action. Thus it has been shown that the first and subsequent rounds of enhancer-dependent transcription in vitro occur with similar rates, suggesting that no ‘‘memory’’ is established during the first round18 (Fig. 3).

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Fig. 2. Analysis of glnAp2 promoter activation by NtrC-dependent enhancer using singleand multiple-round transcription assays. The experimental approach is outlined at the top. (A) Characterization of the enhancer-dependent transcription system in vitro using supercoiled plasmid pTH8 (0.11-kb enhancer–promoter spacing). Different combinations of the purified proteins were analyzed in a single-round transcription assay (lanes 1 to 6). The specific product (309 nucleotide transcript) was generated only in the presence of the full set of proteins (lane 6) and ATP (not shown). Asterisk indicates position of nonspecific transcript that is present when the core RNAP and a plasmid template are added to the reaction.3 Note that specific transcription entirely depends on the presence of each component in the reaction. M, end-labeled pBR322–MspI digest (it was used in all experiments, which include analysis of labeled RNA or DNA). (B) DNA supercoiling is required for activation of transcription over a 2.5-kb distance17 (copyright 2001 National Academy of Sciences, USA). sc plasmid templates having 0.11- or 2.5-kb enhancer–promoter spacing (pLR100 and pLY10 plasmids, respectively17) were incubated in the presence or in the absence of topo I (þ/ topo I); incubation with calf thymus topo I converts DNA into a completely relaxed state. Then transcription was conducted under single7 round (þ heparin) or multiple-round (heparin) conditions. The loading control (227-bp end-labeled DNA fragment) was added to the reaction mixtures immediately after terminating the reaction.

The single-round assay is based on the observation that pre-formed RPo is stable in the presence of heparin (80 g/ml); however, its formation de novo is strongly inhibited.24 First, the components are incubated in the presence of ATP as the sole nucleotide. ATP is required at two points in

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Fig. 3. Quantitative analysis of the rates of single-and multiple-round enhancer-dependent transcription of the glnAp2 promoter18 (copyright 2002 Oxford University Press). The experimental strategy for comparison of the rates of single- and multiple-round transcription is outlined at the top. (A) Time courses of single-round and multiple-round transcription of supercoiled pLR100 plasmid having 110-bp enhancer–promoter spacing. Reaction mixtures were incubated in the presence of all nucleotides (multiple round) or with ACG mixture only (single round). Use of the ACG mixture instead of ATP prevents conversion of the RPo back to RPc24 and thus allows comparison of single- and multiple-round transcription under similar conditions. Labeled transcripts were analyzed in a denaturing PAGE. The loading control (a 227-bp end-labeled DNA fragment) was added to the reaction mixtures immediately after

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the transcription cycle to obtain the open complex. First, ATP is required for the phosphorylation of NtrC by NtrB to form the active form of the activator.7,8 Second, the cleavage of ATP by oligomerized NtrCP bound at the enhancer is required for the formation of the open complex by RNA polymerase.25 In the absence of additional nucleotides, the open complex accumulates. Transcripts are then obtained by adding a mixture of the remaining nucleotides (CTP, GTP, UTP) and heparin, which prevents reinitiation. In some experiments, DNA (that is highly supercoiled after its isolation from bacterial cells) is completely relaxed with calf thymus topo I.17,18 To obtain early elongation complexes [RPel], the components are incubated in the presence of all nucleotides except UTP. During this incubation, open complexes are formed, transcription initiation proceeds, and elongation complexes are stalled at positions requiring the incorporation of UTP (at nucleotide 1818,26). These ternary complexes of template, polymerase, and mRNA are extremely stable; thus, their accumulation serves as a useful reporter of open complex formation. To obtain transcripts, the ternary complexes are then given a mixture of UTP and heparin, allowing for the formation of full-length transcripts and preventing reinitiation. The protocol provided here is most useful for studies of enhancer action and better reflects the situation in vivo, where initiation occurs readily on open complex formation. The multiple-round assay is similar to the single-cycle assays, except that all components are present from the start and heparin is not included. 1. Form initiation complexes in 50 l TB at the following final concentration of the components: 2.8 nM supercoiled (sc) plasmid DNA, 500 nM core RNAP, 1000 nM
54, 120 nM NtrC, 400 nM NtrB. Incubate  at 37 for 15 min to form RPc. Under these conditions, NtrC binds quantitatively to the enhancer and RNAP forms the RPc at the promoter, but enhancer–promoter communication does not occur.3,4,6,9,17,18

24

J. Feng, T. J. Goss, R. A. Bender, and A. J. Ninfa, J. Bacteriol. 177, 5523 (1995). D. S. Weiss, J. Batut, K. E. Klose, J. Keener, and S. Kustu, Cell 67, 155 (1991). 26 Y. Tintut, J. T. Wang, and J. D. Gralla, Genes Dev. 9, 2305 (1995). 25

terminating the reaction. (B) Quantitative analysis of data shown in Fig. 4A. The intensities of the bands containing 484 nucleotide transcripts were quantified using a PhosphorImager. The half-time for transcription initiation in the first round was 1 min and during multiple-round transcription one transcript was synthesized every 2 min. Thus transcription initiation during the first and subsequent rounds occurs with similar rates.

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2. To completely relax plasmid DNA, add topo I to a final  concentration of 0.1 units/l and incubate at 37 for 30 min.  3. Add ATP to a final concentration of 4 mM and incubate at 37 for another 15 min to form RPo. 4. To form RPel, add GTP and CTP to final concentration of 50 M  each and incubate at 37 for 10 min. 5. For single-round transcription, add heparin to a final concentration of 80 g/ml. 6. Add RNase inhibitor to a final concentration of 0.2 unit l, GTP, UTP, and CTP to final concentrations of 80 M together with 2.5 Ci  [-32P]UTP. Incubate at 37 for various times. 7. Add equal volume of 2X TSB to terminate the reaction. 8. Add loading control (a labeled DNA fragment having a distinct mobility in a denaturing gel). 9. Extract the samples with 100 l phenol:chloroform (1:1). 10. Precipitate with ethanol, wash with 70% ethanol, and dissolve in 5 l 100% formamide.  11. Denature the sample at 90 for 1 min, cool on ice, and load 1–3 l on 8% denaturing urea-containing PAGE. Analysis of the Rate of Enhancer–Promoter Communication

The rates of enhancer–promoter communication on supercoiled and relaxed DNA templates can be compared quantitatively using a singleround transcription assay. In this case, communication is initiated by adding ATP after preformation of RPc and NtrC–DNA complex. Because both RNAP and the enhancer-binding protein (NtrC) are prebound to DNA, measurements of the rate of enhancer–promoter communication are not complicated by processes of establishing DNA–protein interactions17 (Fig. 4). The addition of ATP results in NtrC phosphorylation that initiates NtrC–RNAP communication and eventually leads to RPo formation. 1. Form RPc in 50 l of TB as described earlier. Add ATP to a final concentration 0.5 mM to initiate enhancer–promoter communication.  Incubate at 37 for different times (usually from 1 to 30 min). 2. Terminate enhancer–promoter communication and start singleround transcription by adding heparin to a final concentration of 80 g/ml and NTPs to a final concentration of 80 M [together with 2.5 Ci of [-32P]UTP and RNase inhibitor (final concentration 0.2 units/l)].  3. Incubate the samples at 37 for 10 min to complete transcription.

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Fig. 4. Analysis of the rates of enhancer–promoter communication over a large distance on relaxed and supercoiled DNA17 (copyright 2001 National Academy of Sciences, USA). (A) Time courses of enhancer–promoter communication on relaxed and sc plasmids having 0.11- or 2.5-kb enhancer–promoter spacing. Analysis of labeled transcripts in a denaturing PAGE. The experimental strategy for comparison of the rates of enhancer–promoter communication on relaxed (topo Iþ) and sc (topo I) plasmids having 0.11- or 2.5-kb enhancer–promoter spacing is outlined at the top. The control pAN6 plasmid does not contain the enhancer. See text for detail. (B) Quantitative analysis of data shown in A. The intensities of the bands containing 484 and 401 nucleotide transcripts were analyzed using a PhosphorImager. Transcription is saturated after three to four rounds, probably because ATP pool is depleted by NtrC.

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Fig. 5. Analysis of purified RPo and RPel18 (copyright 2002 Oxford University Press). The experimental strategy for purification of RPc and RPo complexes is outlined at the top. (A) RNA polymerase subunit
54 is depleted from the early elongation complex. Complexes were purified from DNA-free proteins on a Sephacryl S-400 column, separated in an SDS–PAGE, and silver stained (lanes 6 and 7). Purified proteins were loaded as additional markers (lanes 1 to 4). Total proteins present in the reaction mixture were stained with Coomassie (lane 5). M, protein molecular mass markers. (B) Functional RPo and RPel complexes survive the Sephacryl S-400 column. RPo and RPel complexes were analyzed using a single-round transcription assay before () or after (þ) fractionation on a Sephacryl S-400 column. No DNA-free proteins were added to the reaction after the column. The majority of the complexes (about 75%) remain functionally active after purification on the column.

4. Add an equal volume of 2XTSB to terminate the reaction. 5. Prepare the samples and run denaturing PAGE as described earlier. Purification of Initiation and Early Elongation Complexes by Gel Filtration

The RPo and RPel formed on plasmid DNA can be purified from proteins that are not bound or loosely bound to the DNA by gel filtration chromatography. Remarkably, both complexes remain functionally active after the chromatography18,27 (Fig. 5B; RPc does not survive the chromatography). This procedure is particularly useful for the analysis of protein composition of the complexes (Fig. 5A). Thus it has been shown that
54 is dissociated from the template after the RNAP leaves the GlnAp2 promoter; this experiment provided structural evidence for the 27

A. J. Ninfa, E. Brodsky, and B. Magasanik, in ‘‘DNA–Protein Interactions in Transcription.’’ A. R. Liss, New York, 1989.

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lack of the ‘‘memory’’ during enhancer action.18 This technique is complementary to the analysis of the complexes by native PAGE and DNase I footprinting.3,4,18,26 1. For the experiments, including purification of RPo and RPel by gelfiltration chromatography, the complexes are prepared as described earlier but in larger amounts (in 200 l) with the following concentrations of the components: 100 nM supercoiled plasmid DNA, 1 M core RNA polymerase, 6 M
54, 1.5 M NtrC, and 1 M NtrB. 2. Equilibrate a 4-ml (14
0.6 cm) Sephacryl S-400 column by washing with 8 ml TB at the rate of 0.2 ml/min. 3. Load 200 l RPo or RPel. Wash the column with 1 ml TB. 4. Elute the complexes with 1 ml TB. Collect eluate in 100-l aliquots. 5. Measure OD260 to identify the DNA peak. The DNA–protein complexes should coelute with plasmid DNA in the void volume while unbound proteins are retained on the column. 6. Pool peak fractions having a DNA concentration of 0.5 A260 or higher. At this point, functional activities of the complexes or their protein composition can be analyzed. 7. Analysis of functional activity: Add all NTPs to a final concentration of 0.2 mM to the pooled fractions to begin (RPo) or resume (RPel) a single-round transcription as described previously. Stop the reaction and analyze the transcripts as described earlier. 8. Analysis of protein composition: Add one-fourth volume of 5X  SPLB and incubate the samples at 95 for 3 min. Separate the proteins in a 10% SDS–PAGE. Stain the gel with the silver stain kit to visualize the proteins (Fig. 5A). Acknowledgment The work was supported in part by NIH Grant GM58650 to V.M.S.