An improved method for the purification of DNA-dependent RNA polymerase from Escherichia coli

Journal of Biochemical and Biophysical Methods, 15 (1988) 235-240 Elsevier

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BBM 00641

An improved method for the purification of DNA-dependent RNA polymerase from Escherichia coli K. Prasanna Kumar and D. Chatterji Centre for Cellular and Molecular Biology, Hyderabaa~ India (Received 22 September 1987) (Accepted 29 September 1987)

Summary DNA-dependent RNA polymerase from Escherichia coli was purified further by elution through heparin-Sepharose CL-6B column after the enzyme was obtained, partially purified, using Burgess and Jendrisak's method [(1975)Biochemistry 14, 4634] The total yield of the pure protein was 10 mg from 50 g of E.coli cells. The method was found to be very reproducible and convenient. The enzyme preparation had 60% active molecules and the elongation rate of RNA synthesis by this enzyme was measured to be 11 bases/s over ADll 1 T7 DNA. Key words: Purification; RNA polymerase, E.coli; Heparin-Sepharose column

Introduction T h e p r i m a r y event in the expression of genes is the transfer of genetic i n f o r m a tion from D N A to R N A molecules with the help o f the e n z y m e k n o w n as D N A d e p e n d e n t R N A p o l y m e r a s e (Rpase, N u c l e o t i d e t r i p h o s p h a t e : R N A n u c l e o t i d y l transferase E C 2.7, 7.6). F r o m a b a c t e r i a l source such as Escherichia coli this e n z y m e has b e e n p u r i f i e d to h o m o g e n e i t y p r e v i o u s l y [ 1 - 3 ] a n d all the c o n d i t i o n s for the specific t r a n s c r i p t i o n in vitro have b e e n established. It is a large p r o t e i n ( M W . 500 k D a ) w i t h five s u b u n i t s a 2 flfl'o. T h e o s u b u n i t is the one which confers o n the core R p a s e ( a 2 f l f l ' ) its a b i h t y to initiate the specific t r a n s c r i p t i o n . D u r i n g o u r i n v e s t i g a t i o n on the role o f the s i g m a s u b u n i t d u r i n g the i n i t i a t i o n of t r a n s c r i p t i o n , we o b s e r v e d that a n y c o n t a m i n a n t p r e s e n t in the h o l o - e n z y m e p r e p a r a t i o n usually

Correspondence address: D. Chatterji, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, 500 007 (A.P.), India. 0165-022X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

236 carries over to the sigma unit at the time of its dissociation from the holo-enzyme by the conventional methods [3,4]. This has made the isolation of the pure sigma difficult. Moreover, all the methods so far known to purify Rpase free of contaminants either employ phosphocellulose chromatography at high glycerol concentrations, which is time consuming with other practical difficulties [2], or start with a large quantity of biomass of the bacteria [3]. We report here the isolation of pure E.coli Rpase from 50 g of the biomass where the yield of the final product is appropriate (10 mg), the method convenient, reproducible and just a one-step addition to the usual Burgess method [1].

Materials and Methods

All chemicals purchased and used in this study are of the highest purity available. [3H]UTP or [a-32p]UTP were from Amersham U.K. Nucleotides and calf thymus DNA were purchased from Sigma Chemical Co. ADmT 7 DNA was a gift from C.W. Wu of SUNY, Stony Brook, New York. RNA polymerase was purified from 50 g of E.coli cells MRE600 (RNaseI-) grown in enriched (LB) media upto 3/4th of log phase, following the protocol of Burgess and Jendrisak [1]. For this purpose all the column sizes, volume of buffers, polymin P fractionation steps and other parameters were adjusted, as the original method describes the protocol for a large quantity of starting material. The active fractions thus obtained from the eluate of the bio-gel column were found to contain 14 mg of protein. 5 g of heparin-Sepharose CL-6B (Pharmacia) were suspended in 15 ml of buffer I containing Tris-HC1, pH 8, 10 mM MgC12, 1 mM EDTA, 0.3 mM dithiothreitol (DTT) and 7.5% glycerol. The process was repeated several times till the heparinSepharose was well equilibrated with buffer I. RNA polymerase was dialysed against buffer containing 10 mM Tris-HC1, pH 8, 10 mM MgC12, 0.1 mM EDTA and 0.1 mM DTT and 5% glycerol with 0.5 M NaC1. Equal volumes of heparin-sepharose and the enzyme (14 mg) were mixed together and agitated in the cold-room for 3 h. The whole slurry was subsequently packed in a column (15 ml) and washed with buffer I + 0.3 M NaC1. Subsequently the active fractions of the Rpase were eluted with buffer I + 0.6 M NaC1 at a flow rate of 0.2 ml/min. Recovery of the enzyme from heparin column was found to be about 10 mg. SDS-PAGE analysis of the product [5] showed 60-70% sigma saturation in the holo-enzyme preparation. The enzyme was assayed over calf thymus o r A D l l I T 7 DNA following the method of Lowe et al. [3].

Results

Fig. 1 shows the elution profile of the Rpase from the heparin-Sepharose column. Table 1 compares the specific activity of the bio-gel enzyme and the enzyme obtained after heparin column. We have noticed that the enzyme obtained after the Burgess and Jendrisak procedure [1] routinely contained many impurities. This is

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Fig. 1. Heparin-Sepharose column chromatography of E.coli R N A polymerase (see text for details).

probably because we started with less biomass of E.coli than that was recommended in the original procedure. However, the improvement in the purification was remarkable, as also seen from Fig. 2. Following the protocol of Chamberlin et al. [6], we have also calculated the number of active Rpase molecules in our preparation. Fig. 3 shows the in vitro RNA synthesis of A D m T 7 DNA, which has a single strong promoter (T7A1) that gives rise to a discrete RNA of 6100 nucleotides [7]. The transcription was carried out in the presence of heparin to block reinitiation. As it can be seen from the figure, within 11 min one round of RNA synthesis is coming to an end whereas the

TABLE I C O M P A R I S O N O F T H E Y I E L D A N D T H E ACTIVITY O F E.COLI R N A P O L Y M E R A S E OBT A I N E D F R O M BURGESS'S M E T H O D [1] A N D F U R T H E R E L U T I O N T H R O U G H H E P A R I N SEPHAROSE COLUMN Step

Total protein

Specific activity *

Bio-Gel A 1.5 M (Last step of Ref. 1) Heparin-Sepharose CL-6B

14.4 mg 10.0 m g

583 2272

* Specific activity is defined as nmole of [3HI or [a-32p] U T P incorporated into DE-81 filter bound counts per m g of protein per 20 rain at 37 o C. Total starting material was 50 g of wet weight E.coli cells.

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Fig. 2. Sodium dodecylsulfate-polyacrylamide(10%)gel electrophoresisof E.coli RNA polymerase.Lane 1; protein (38 #gin) after bio-gel A 1.5 M column, Lane 2 and 3; different batches of proteins (18 #gin) eluted from bio-gelA 1.5M after passing through heparin-Sepharosecolumn. initial lag of the start of the synthesis is about 20 s. From Fig. 3 and the calculations described in detail before [6], it was found that the heparin-Sepharose eluate enzyme preparation had about 60% active molecules and the rate of elongation of the R N A chain by this enzyme was 11 bases/s.

Discussion So far, the best method [2] available for the homogeneous purification of E.coli Rpase is the phosphocellulose chromatography of the enzyme obtained from the Burgess procedure [1]. This involves elution of the column with a buffer containing 50% glycerol in order to prevent the dissociation of the sigma subunit from the enzyme. However, some dissociation cannot be avoided; also, the presence of high amounts of glycerol makes the elution troublesome. Chamberlin et al. [8] have described the use of heparin-agarose column for the one-step purification of RNA polymerase from one gram of cells. Although this enzyme is good for routine transcription analysis, the presence of other contamina-

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Fig. 3. Heparin resistant RNA synthesis over ADII1T7 DNA by purified E.coli RNA polymerase. Heparin was added after 2 min of initiation of RNA synthesis according to the protocol described in [6]. 'Ti' denotes the time at which all of the RNA polymerase molecules in the reaction complete one round of RNA synthesis and ' To' is the time period required for template binding and RNA chain initiation.

tion with the e n z y m e c a n n o t b e ruled o u t and, f r o m such a p r e p a r a t i o n , the i s o l a t i o n o f the p u r e sigma s u b u n i t is very difficult. M o r e o v e r , h e p a r i n - a g a r o s e is n o t available c o m m e r c i a l l y unlike h e p a r i n - S e p h a r o s e C1-6B a n d o n e has to p r e p a r e the m a t e r i a l f r o m C N B r - a c t i v a t e d a g a r o s e [9]. It thus a p p e a r s that, in c o m p a r i s o n to all o t h e r procedures, use o f h e p a r i n - S e p h a r o s e for the p u r i f i c a t i o n o f E.coli R N A p o l y m e r a s e is m u c h m o r e c o n v e n i e n t a n d far m o r e r e p r o d u c i b l e .

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Simplified description of the method and its application We have reported here further purification of Escherichia coli D N A - d e p e n d e n t R N A polymerase after the enzyme was obtained in a partially pure f o r m following Burgess and Jendrisak's protocol [(1975) Biochemistry 14, 4634]. T h e active fractions were eluted through heparin-Sepharose CL-6B in buffer containing Tris ( p H 8), 10 m M MgC12, 1 m M E D T A , 0.3 m M dithiothreitol, 7.5% glycerol and 0.6 M NaC1. 10 m g of pure enzyme was obtained f r o m 50 g of E. coli biomass. The enzyme preparation had 60% of the active molecules.

References 1 2 3 4 5 6 7 8

Burgess, R.R. and Jendrisak, F.F. (1975) Biochemistry 14, 4634-4638 Gonzalez, N., Wiggs, T. and Chamberlin, M.J. (1977) Arch. Biochem. Biophys. 182, 404-408 Lowe, P.A., Hager, D.A. and Burgess, R.R. (1979) Biochemistry 18, 1344-1352 Burgess, R.R. and Travers, A.A. (1971) Methods Enzymol. 21,500-506 Laemmli, U.K. (1970) Nature (London) 227, 680-685 Chamberlin, M., Nierman, W., Wiggs, T. and Neff, N. (1979) J. Biol. Chem. 254, 10061-10069 Wiggs, J., Bush, J. and Chamberlin, M. (1979) Cell 16, 96-109 Chamberlin, M., Kingston, R., Gilman, M., Wiggs, J. and De Vera, A. (1983) Methods Enzymol. 101, 540-568 9 Davison, B., Leighton, T. and Rabinowitz, J.C. (1979) J. Biol. Chem. 254, 9220-9226