Eur. J. Biochem. 60, 51 - 55 (1975)

Rapid Isolation of Highly Active RNA Polymerase from Escherichia coli and Its Subunits by Matrix-Bound Heparin

Hans STERNBACH, Reinhild ENGELHARDT, and A. G. LEZIUS

Max-Planck-Institut fur Experimentelle Medizin, Abteilung Chemie, Gottingen

(Received June 4/August 29, 1975)

1. RNA polymerase from Escherichia coli is selectively and strongly retained by a heparin-sub- stituted agarose and can be eluted therefrom by a neutral buffer containing 0.6 M salt. The method is applicable to relatively crude preparations of the enzyme on a preparative scale giving highly purified RNA polymerase in excellent yield. The enzyme obtained by this procedure shows the highest specific activity so far reported and is pure and enriched in factor CJ as indicated by dodecylsulfate gel electrophoresis.

2. Based on the differential affinity of the subunits of the enzyme for the heparin-carrying gel matrix, a method for separation of CI, p’ + p and 0 subunits by application of urea and salt-containing buffers is described. Upon recombination and dialysis with urea-free buffer 40- 50 % of the enzyme activity is restored.

The linear acid mucopolysaccharide heparin is able to interact strongly with several proteins by virtue of its special surface charge distribution. Probably by competition with nucleic acids [l], the polymer inhibits ribonucleases [2 - 41 and bacterial and eukaryotic DNA-dependent RNA polymerases, the latter by interfering with the initiation of transcrip- tion [4-61. In one case, the affinity of an enzyme for heparin has been exploited for its purification : lipo- protein lipase was isolated by chromatography on heparin-substituted agarose [7]. It therefore seemed worthwhile to us to explore this method for the isola- tion of RNA polymerase and for studying the affinity of this enzyme to matrix-bound heparin.

EXPERIMENTAL PROCEDURES

Materials

Escherichia coli strain MRE 600 was purchased from E. Merck (Darmstadt, Germany). Calf thymus DNA and poly[d(A-T)] sodium salt were products from Boehringer, Mannheim (Germany), deoxyribo- nuclease I (3750 units/mg, RNAse-free) from Worth- ington Biochem. Corp. (Freehold, New Jersey,

Enzymes. RNA polymerase (EC 2.7.7.6); DNAase I (EC 3.1.4.5).

U.S.A.). All other chemicals used were of the highest purity available from commercial sources.

Preparation of Heparin-Sepharose

Heparin (Vetre@’, 125 IU/nig, U.S.P. XVII, Pro- monta Hamburg, Germany) was covalently bound to agarose gel (Sepharose 4B, Pharmacia Fine Chemicals, Uppsala, Sweden), activated by treatment with cyano- gen bromide [8]. Bound heparin was determined by sul- fate analysis after hydrolysis of a lyophilized sample of heparin-Sepharose by the method of Antonopoulos [9]. The sulfate content of heparin-Sepharose 4B (20.8 pg/ mg dry weight) had to be corrected for the blank given by unmodified Sepharose 4B (2.56 pg/mg dry weight). The difference, 18.24 pg sulfate/mg dry weight corresponds to 4.35 pg bound heparinlmg dry weight, based on the molecular weight of 14600, as taken from the literature [lo]. Since 10.5 mg lyophilized heparin- Sepharose give 1 ml wet gel, as determined indepen- dently by Iverius [8], there are 45 pg/ml heparin bound. Before use the heparin-Sepharose was ex- haustively washed with buffer A (0.01 M Tris/acetate pH 7.5, 0.01 M magnesium acetate, 1.0 mM dithio- threitol) containing 1.0 M NH4C1 and finally equi- librated with buffer A + 0.2 M NH4Cl. Heparin- Sepharose can be used several times without loss of

52 Isolation of RNA Polymerase by Matrix-Bound Heparin

Fig. 1. Elution diagram of RNApolyrnerase from a heparin-Sephauose column. (-0) Absorbance at 280 nm; ( x ~ x ) RNA polymerase activity; (-) molarity of NH,Cl. 5-ml fractions were collected at a flow rate of about 20 ml/h. Fractions 47 - 63 were pooled and concen- trated

capacity if washed exhaustively with buffer A contain- ing 1 .O M NH,Cl and 6.0 M urea to remove any bound protein.

Chromatography of RNA Polymerase on Heparin-Sepharose

Crude RNA polymerase was prepared from 500 g E. coli cells according to the procedure of Burgess [ll] up to step 3. The twice-washed ammonium sulfate pellet was dialyzed overnight against buffer A contain- ing 0.2 M NH,Cl. The dialyzate was diluted with the same buffer to obtain a protein concentration of about 30 mg/ml. The protein concentration was determined by measuring the absorbance at 280 nm and 260 nm according to Warburg et al. [12]. The solution (150 ml) was applied to a 2 x 12-cm column of heparin- Sepharose which was equilibrated with buffer A containing 0.2 M NH,Cl. Subsequently the column was washed with buffer A + 0.3 M NH,C1 until the absorbance at 280 nm was lower than 0.1. A linear salt gradient was applied from buffer A + 0.3 M NH4C1 to buffer A + 0.8 M NH,C1 (total volume of 500 ml). The fractions containing the majority of the enzyme activity were pooled. RNA polymerase was assayed according to Burgess [ll]. One unit of RNA polymerase is defined as the amount of enzyme able to incorporate one nanomole of [14C]AMP in ten minutes at 37 "C under the given conditions. In order to concentrate the enzyme it was bound again to heparin-Sepharose after dilution with buffer A to a final concentration of about 0.3 M NH,CI. The enzyme was eluted again with a small amount of buffer A containing 0.7 M NH,Cl. For storage at -20 "C an equal volume of glycerol was added to the enzyme solution.

Fractionation of the RNA Polymerase Subunits

For fractionation of the different subunits of RNA polymerase, 10 mg of the pure enzyme was bound on 3 ml heparin-Sepharose. The column (1 cm diameter) was washed with increasing amounts of urea in buffer A and finally with urea in the presence of high-ionic-strength buffer. The separated protein- containing fractions were dialyzed against buffer A + 0.5 M NH,C1 to remove the urea and precipitated by addition of saturated ammonium sulfate solution in buffer A to give a 60 % saturated solution. The precipitates were centrifuged for 20 min at 15 000 rev./min and the resulting pellets dissolved in a small amount of buffer A. An equal volume of glycerol was added to each solution for storage at -20 "C.

Sodium Dodecyl Sulfate Gel Electrophoresis

Dodecylsulfate gel electrophoresis of the enzyme and its subunits was performed in 5 % gels as described by Shapiro et al. [13]. Gels of 9.5-cm length were used. After staining the gels were scanned with a Vitatron Densitometer TLD 100.

RESULTS

Pur fication of RNA Polymerase

Agarose gel containing covalently bound heparin binds quantitatively E. coli RNA polymerase from crude preparations. The holo enzyme can be eluted by a linear salt gradient. The maximum of the RNA polymerase activity elutes between 0.56- 0.60 M NH,C1 (Fig. 1). By this single step an 80-fold purifica- tion of the enzyme activity is obtained. A summary of the purification of RNA polymerase from 500g

H. Sternbach, R. Engelhardt, and A. G. Lezius 53

Table 1. Summary of RNA polymerase purification

Purification step Total protein Total activity Specific activity A**, nm A260 "In

mg Ammonium sulfate fractionation after

dialysis 4 650 Heparin-Sepharose chromatography 122 High-salt/sucrose gradient centrifugation 108

U

110000" 240 000 212000

U/mg protein

24 1970 1960

1.04 1.58 1.92

a Due to the presence of DNAase I and of the large amount of polynucleotides [ I l l the total activity is not very meaningful.

E. coli cells is given in Table 1. The capacity of the heparin-Sepharose prepared is very high for pure holo RNA polymerase. 1 ml of wet gel with 45 pg heparin coupled is able to bind up to lOmg of enzyme. Using an average molecular weight of 14600 for hepa- rin and 500000 for the holo enzyme (p ' , p, ci, 2a) 1 mol of heparin binds about 6 mol of RNA poly- merase.

Dodecylsulfate gel electrophoresis (Fig. 2) of the denatured enzyme reveals the normal subunit pattern of the four different polypeptide chains p', p, CJ and a. In contrast to normal RNA polymerase preparations the content of factor CJ is markedly higher, which may be one reason for the considerably increased specific activity of the enzyme purified on heparin- Sepharose. Besides the normal subunits there are two minor bands designated by X and o (Fig. 2). The relative mobility of these two proteins compared with p', p, a and ci yields molecular weights of 11 5 000 and 11 000 respectively. The A280/A260 ratio of this enzyme preparation is less than 1.6 due to a certain content of nucleic acid which binds to the enzyme at low ionic strength. Probably these nucleic acid fragments are protected by the enzyme molecule and escaped the preceding DNAse treatment. They can be removed by means of a glycerol or sucrose gradient centrifuga- tion in presence of high-ionic-strength buffer [ l l]. A ratio of A280/A260 > 1.8 indicates that the enzyme is free of any nucleic acid contamination.

Isolation of the Different Subunits of RNA Polymerase

Desorption with urea of pure holo RNA polymer- ase obtained by heparin-Sepharose chromatography demonstrates differing affinity of the individual sub- units of the holo enzyme for the matrix-bound heparin. Washing a column loaded with RNA poly- merase with 2.0 M urea in buffer A releases protein which shows three distinct bands in dodecylsulfate gel electrophoresis. The main components seem to be identical with the 'X'-protein of other authors [14- 161 and the factor cc) [17] (Fig. 2 densitogram 2). The minor component is a small proportion of the a subunit which can be eluted completely and without

t

- Migration

Fig. 2. Dodecylsuljate gel electrophoresis oJ R N A polymerase ( I ) and of R N A polymerase subunits (2-5) after rechromatography. (A) Photographs of the gels; (B) densitograms of the gels. 1 RNA polymerase obtained by heparin-Sepharose chromatography; 13 pg of protein were applied to the gel. 2 Proteins eluted from heparin- Sepharose with buffer A + 2.0 M urea (4.8 pg). 3 Subunit c[ ob- tained by elution from heparin-Sepharose with buffer A + 4.0 M urea (7.2 pg). 4 Proteins eluted with buffer A + 4.0 M urea + 0.4 M NH,CI from heparin-Sepharose (8.6 pg). 5 Factor 0 after elution from heparin-Sepharose with buffer A + 6.0 M urea + 1.0 M NH,C1(9.9 pg)

any contamination using 4.0 M urea in buffer A (Fig. 2 densitogram 3) . By further increasing the urea concentration to 7.0 M no more protein can be washed out from heparin-Sepharose. Increase of ionic strength

54 Isolation of RNA Polymerase by Matrix-Bound Heparin

in the presence of urea (buffer A + 4.0 M urea + 0.4 M NH,Cl) yields the subunits p’ and f l which are contaminated with a trace of factor cr. In addition there appears a protein with an extremely high molec- ular weight (Fig. 2 densitogram 4). Since f l ’ , p, cr and CY can be separated from each other by dodecyl- sulfate gel electrophoresis without treatment with reducing agents (2-mercaptoethanol) [17], this heavy protein is probably an aggregate of p’ or /3 or a co- aggregate of both. Factor (T shows the highest affinity to heparin-Sepharose. It is eluted with bufferA + 6.0 M urea + 1.0 M NH,Cl (Fig. 2 densitogram 5). Rechromatography of the isolated factor cr on heparin- Sepharose shows that factor cr possesses this high affinity to matrix-bound heparin as an intrinsic property. Also CI and p‘ + p can be bound separately to heparin-Sepharose, and are eluted with buffer A + 4.0 M urea and buffer A + 4.0 M urea + 0.4 M NH,Cl, respectively. This indicates that the different affinity of the separated subunits is independent of the presence of the other RNA polymerase subunits.

The isolated subunits can be stored for several weeks in 50 % glycerol at -20 “C. After reassembly in the appropriate molar ratio, the reconstituted RNA polymerase molecule shows 40 - 50 % of the original activity after preincubation. The isolated subunits themselves are completely inactive. Table 2 summari- zes the activity of the subunits during stepwise re- assembly in comparison with the activity of the original holo enzyme using calf thymus DNA and poly[d(A-T)] as template. Factor cr stimulates the incorporation of [14C]AMP with DNA as template in presence of CTP, GTP and UTP markedly while the stimulation of the translation of poly[d(A-T)] is insignificant. Addition of the ‘X’-protein and factor w effects a further stimulation of the AMP incorporation by about 30% when DNA as template is used.

DISCUSSION

Heparin is a very potent and specific inhibitor of RNA polymerases in that it interferes with the initia- tion of transcription. Therefore the term affinity chromatography is justified to describe the binding of the enzyme to the immobilised inhibitor. Affinity chromatography of E. coli RNA polymerase on matrix-bound heparin provides the holo enzyme in high yield. This preparation is twice as active as other enzyme preparations. The higher specific activity may have several reasons. First of all we believe that the unusually high content of factor cr of the enzyme purified on heparin-Sepharose is responsible for the increased specific activity. RNA polymerase which is prepared by a common procedure shows 30- 50 % deficiency of factor cr in comparison to the other sub-

Table 2. Activity of RNA polymerase and its subunits 30 prnol of each subunit was used

Subunits Incorporation of [‘4C]AMP with

8‘ + B 0 0 B’ + B + 2a 0.89 37.8 8’ + 8 f 2U + G 10.8 41.5 p’ + p + 2a + G + ‘X’ + w 46.6 Enzyme purified :

on heparin-Sepharose 33.5 98.5

14.1

after Burgess [ 11,171 17.4 49.5

units [18]. By addition of factor (T to such an enzyme the translation of native DNA can be stimulated very effectively. Furthermore probably enzymically inactive RNA polymerase does not bind tightly to heparin-Sepharose as was shown with rifampicin- Sepharose by other authors [16]. Lastly heparin also inhibits ribonucleases. These may also have a pro- nounced but different affinity for the heparin-sepha- rose and can thus be removed quantitatively. Degrada- tion of the reaction products of RNA polymerase is therefore very unlikely.

Calculation of the binding capacity of the prepared heparin-Sepharose based on the data employed by Walter et al. [ 5 ] (heparin, M , = 12500, holo RNA polymerase, M , = 720000) suggests the binding of 4 mol of enzyme to 1 mol of matrix-bound heparin. In contrast, these authors reported that two heparin molecules are required for the inactivation of one holo enzyme molecule. This discrepancy between binding capacity and inactivation cannot be explained at present with the data given in the literature and our own results.

The strong binding of RNA polymerase to matrix- bound heparin may be explained to some extent by the fact that at least two subunits are firmly attached to the inhibitor. One site of attachment involves p’ as was argued by Walter et al. [5]. A second binding site on the holo enzyme must be present on the cr sub- unit. Factor cr, which shows a very weak binding to phosphocellulose is retained most strongly by heparin- Sepharose. It therefore seems that not only the overall difference of charges between protein and polyanion but also, and even more importantly, the distribution of the charges is responsible. Factor cr itself can only be eluted from heparin-Sepharose using 6 M urea + 1.0 M NH4Cl whereas factor cr in conjunction with the core enzyme is eluted at lower ionic strength (0.6 M NaCl) without urea. Because of its high affinity factor cr may be specifically responsible for

H. Sternbach, R. Engelhardt, and A. G. Lezius 55

the inhibition of initiation by heparin, possibly by removal of G from the complex holo-enzyme . DNA.

The a or a2 subunit clearly shows the lowest affinity to heparin-Sepharose under conditions of denaturation, as it is eluted at urea concentrations sufficient to dissociate the enzyme. We have been unable to separate p’ and /3 up to the present using this method although they are completely different with regard to their amino acid sequence and their basicity. Therefore we envisage a binding of the subunit to the heparin-Sepharose via a specific inter- action of p with p‘. The breakdown of this interaction leads to an irreversible denaturation of p’ and p [19].

The advantages of this new procedure to purify RNA polymerase holo enzyme as well as the subunits are obvious. We are convinced that it will shortly find broad application not only for the isolation of RNA polymerase from E. coli but also from other organisms. Preliminary experiments using mammalian tissue have given similar results. Besides these aspects, it opens new possibilities for studying the mode of heparin inhibition.

The authors wish to thank Dr F.vonderHaar for many valuable discussions; they further thank Drs D. Gauss and J. Hobbs for critically reviewing the manuscript and Prof. F. Cramer for his encouraging interest and support.

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H. Sternbach, R. Engelhardt, and A. G. Lezius, Abteilung Chemie, Max-Planck-Institut fur Experirnentelie Medizin, D-3400 Gottingen, Hermann-Rein-StraBe 3, Federal Republic of Germany