[43] Purification of two forms of Escherichia coli RNA polymerase and of sigma component

506

FACTORS AFFECTING GENE EXP R ES S IO N

[43]

mide gels containing 0.1~ SDS, pH 7.2, and containing 8 M urea, pH 8.7, it is not known whether sigma is a single species or whether it is a mixture of several different polypeptide chains with similar properties. Sigma forms a tight, but reversible, 8° complex with core enzyme, even at high salt concentrations, such as 1.0M KC1. Krakow et al. 31 have shown that for Azotobacter vinelandii, the initiation factor is released when the complete enzyme binds to single-stranded polynucleotides. The release of sigma on passage through a phosphocellulose column might be explained by the resemblance between phosphocellulose and such polyanionic substances. Function of Sigma It has been shown that the ability of sigma to stimulate the reading of T4 phage DNA and other DNA's by core enzyme is due to its ability to stimulate initiation of RNA synthesis and not to the stimulation of the rate of chain elongation. 29 During or very soon after initiation, sigma is released from the core enzyme and can form a complex with another core, thus allowing a second enzyme to initiate. In this way sigma acts catalytically to promote initiation. It is not known how sigma stimulates initiation, but it appears likely that it is important in the recognition of specific initiation sites on the DNA. ~°A A. Travers, unpublished result. slJ. S. Krakow, K. Daley, and M. Karstadt, Proc. Nat. Acad. Sci. U.S. 62, 432 (1969).

[ 4 3 ] P u r i f i c a t i o n o f T w o F o r m s of E s c h e r i c h i a coli R N A Polymerase B y D.

a n d of S i g m a C o m p o n e n t

BERG,K. BARRETT,and M. CHAMBERLIN

RNA polymerase holoenzyme ~- core polymerase ~ sigma component Escherichia coli RNA polymerase isolated by most procedures contains a tightly bound subunit, sigma, which is required for specific RNA synthesis. The polymerase holoenzyme can be separated into a core polymerase and sigma component. Procedures are described for the isolation of the RNA polymerase holoenzyme, core polymerase and sigma component, and for reconstitution of the holoenzyme from the separated components.

[43]

PURIFICATION OF RNA POLYMERASE

507

Assay Methods Principle. RNA polymerase activity is determined by following the DNA dependent conversion of the AMP moiety from ATP into an acidinsoluble form. RNA polymerase holoenzyme is assayed with T2 DNA or dAT copolymer as template. The core polymerase shows little or no activity with T2 DNA as template 1,~ and is assayed using dAT copolymer. The activity of sigma component is determined by its ability to stimulate RNA synthesis by the core polymerase when T2 DNA is used as template. Reagents Assay solution A: 0.2M Tris.HC1, pH 8.0, 50 mM MgC12, and 50 mM fl-mereaptoethanol Enzyme diluent: 10 mM Tris.HC1, pH 8.0, 1 mM MgCl~, 1 mM /3-mercaptoethanol, 50 /~M EDTA, and 0.1 mg/ml bovine serum albumin (BSA) Templates: T2 DNA ~ and dAT copolymer4 are prepared as 1 mM solutions in 10 mM Tris.HC1, pH 8.0, with 50 mM NaC1. Polynucleotide concentrations are given in terms of total nucleotide. Substrates: Commercial preparations of unlabeled ATP, UTP, CTP, and GTP are used and are conveniently prepared as 4 mM solutions. [a-32p]ATP with a specific activity of 2000-20,000 cpm/ m/zmole is purchased commercially or is synthesized according to Symons2 Any one of the nucleoside triphosphates may be used as label, and 3H or 14C nucleosides may be substituted for [~-~P]ATP. Procedure Assay for R N A Polymerase Holoenzyme. Assay tubes contain 0.02 ml of assay solution A, 15-20 mttmoles of T2 DNA, 40 m~moles each of CTP, GTP, UTP, and [32p]ATP, and 0.3-3 T2 units of enzyme. The final volume is 0.1 ml. Synthesis is initiated by adding enzyme and is terminated after 10 minutes at 37 ° with 3 ml of ice cold 3.5% perchloric acid containing 0.1 M sodium pyrophosphate. The resulting precipitate is dis1R. R. Burgess, A. A. Travers, J. J. Dunn, and E. K. F. Bautz, Nature (London) 221, 43 (1969). D. Berg, K. Barrett, D. Hinkle, J. McGrath, and M. Chamberlin, Fed. Proc., Fed. Amer. Soc. Exp. Biol. 28, 659 (1969). C. A. Thomas, Jr. and J. Abelson, in "Procedures in Nucleic Acid Research" (G. L. Cantoni and D. R. Davies, eds.), p. 553. Harper and Row, New York, 1966. 4H. K. Schachman, J. Adler, C. M. Radding, I. R. Lehman, and A. Kornberg, J. Biol. Chem. 9.35, 3242 (1960). R. H. Symons, Biochim. Biophys. AcLa 155, 609 (1968).

508

FACTORS AFFECTING GENE EXPRESSION

[43]

persed with a glass pestle and the tubes are chilled on ice for 5--10 minutes. The precipitates are collected on Whatman G F / C filters and are washed with 4 or 5 3-ml aliquots of cold 1 M HC1 containing 0.1 M sodium pyrophosphate and finally with 3 ml of cold ethanol. The radioactivity on the dry filter is determined using a gas flow counter or in a toluene based solvent in a scintillation counter. Assay ]or Core Polymerase. The same procedure is used as described for RNA polymerase holoenzyme except that dAT copolymer (20 m~moles) replaces T2 DNA as template, and CTP and GTP are omitted. From 0.3 to 3 dAT units of enzyme are added to each assay. Assay for Sigma Component. The assay solutions are identical to those described above in the assay for RNA polymerase holoenzyme except that 20-30 dAT units of core polymerase and 0.3--2.0 units of sigma component are added to each assay. Assay tubes are chilled on ice prior to addition of the core polymerase and appropriate aliquots of sigma component are added. Synthesis is initiated by immersing the tubes in a water bath at 37 °. After 10 minutes at 37 ° the reaction is terminated with cold perchloric acid and samples are assayed for acidinsoluble radioactivity as described above. The increase in the rate of AMP incorporation is directly proportional to the amount of sigma component added to the assay over the range of sigma concentration specified. Definition of Units. One unit of RNA polymerase activity catalyzes the incorporation of 1 m~mole of AMP in 1 hour using the standard assays described above. To identify the template used in the assay, units are referred to as dAT units or T2 units when dAT copolymer or T2 DNA, respectively, are used as templates. The activity of sigma component preparations is defined as the increase in the rate of T2 RNA synthesis brought about by addition of a given amount of sigma component to 20-30 dAT units of core polymerase. Thus 1 unit of sigma activity increases the rate of incorporation of AMP by 1 m~mole per hour in a standard assay. Specific activities are expressed as units per milligram of protein. Protein is determined by the method of Lowry et al2 using bovine serum albumin as a standard. Purification Procedures

Reagents and Materials Buffer A: 50 mM Tris.HC1, pH 8.0, 10 mM MgC12, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 5% glycerol 60. Lowry, J. S. Rosebrough, A. C. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

[43]

PURIFICATION OF RNA POLYMERASE

509

Buffer B: 20 mM Tris.HC1, pH 8.0, 10 mM MgC12, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 5% glycerol Buffer C: 50 mM Tris.HC1, pH 8.0, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 5% glycerol Saturated ammonium sulfate solution is saturated at 25 ° with enzyme grade ammonium sulfate (Mann Company) and is then adjusted to pH 7.5-8 with dilute NaOH. It contains 4.1 M ammonium sulfate. Storage solution contains buffer B with 60% (v/v) glycerol DNase solution contains 1 mg/ml of pancreatic DNase 1 (Worthington Biochemical Company, D P 8HD) dissolved in ice cold buffer A. It is prepared immediately before use. Protamine sulfate solution (1%) contains 10 mgfml of protamine sulfate (Lilly Company) in distilled water. It is kept at 37 ° before use. DEAE-cellulose (0.72 meq/g, Bio-Rad Laboratories) is washed and equilibrated with 50 mM (NH4)2C0~ -~ 0.1 mM EDTA Biogel P-300 (50-150 mesh, Bio-Rad Laboratories) is equilibrated with buffer B containing 0.2 M KC1. Phosph0cellulose (P-11 cellulose phosphate, 7.4 meq/g, Whatman Company) is washed and equilibrated with buffer C. Bacterial Cells. E. coli are grown in minimaV or enriched 8 medium; either strain K or B/1 has been used. In late exponential growth phase the cell suspension is chilled with ice and the cells are harvested by continuous flow centrifugation. The cell paste is stored frozen at - - 1 5 ° and has been stored for at least 1 year without loss in activity. Unless otherwise noted the following steps are carried out at 0-4 ° and all centrifugations are performed at 15,000 rpm for 30 minutes with a SS-34 rotor in a Sorvall RC 2B centrifuge. Purification o] R N A Polymerase Step 1. Cell Extract. One hundred grams of frozen cells (E. coli B / I ) are mixed in a Waring Blendor with 300 g of glass beads and 90 ml of buffer A. The speed of the blender is controlled by a rheostat (Powerstat). The mixture is blended at high speed (setting of 75/100). Additional beads are added to the mixture during blending if necessary to keep the consistency of the mixture that of a thick, semisolid slurry. 'M. Chamberlin and P. Berg, Proc. Nat. Acad. Sci. U.S. 48~ 81 (1962). 8A minimal salts medium has been used which was supplemented with 10 g of bactotryptone per liter and 1 g of yeast extract per liter.

510

FACTORS AFFECTING GENE EXPRESSION

[43]

The temperature is kept at 4°-10 ° by periodic cooling of the blender in a dry ice-isopropanol bath. After 12 minutes of blending, 120 ml of buffer A are added and the blender is run for 1 minute at reduced speed (35/ 100). The beads are allowed to settle for 20 minutes, and the supernatant fluid is decanted. T h e beads are then rinsed with 105 ml of buffer A. The supernatant fluid and rinse fraction are combined and this fraction is centrifuged for 90 minutes; the resultant clear supernatant fluid is collected (fraction 1, 185 ml). Step 2. Ammonium Sulfate Fractionation. To fraction I (185 ml) is added 1 ml of DNase solution; the mixture is stirred gently for 5 hours. The speed of stirring is increased, and 66 g of powdered ammonium sulfate is added together with 0.7 ml of 1 M NaOH. After the salt has dissolved, the solution is stirred gently for 20 minutes. The precipitate is collected by centrifugation and is suspended in 150 ml of buffer A containing 2 . 5 M ammonium sulfate. After 20 minutes the suspension is centrifuged. The precipitate is collected and is drained carefully to remove as much salt solution as possible. The precipitate is dissolved in buffer A and is then diluted to give a salt concentration such that a 1:5000 dilution of an aliquot in deionized water reads 2 ppm or less with a standard conductivity meter. (2 ppm corresponds to an ammonium sulfate concentration of 40 m M ) . The final volume obtained is about 480 ml (fraction II). Step 3. Protamine Sulfate Fractionation. To 480 ml of fraction II is added 19.5 ml of a 1% solution of protamine sulfate3 The solution is stirred gently for 30 minutes, then the precipitate is collected by centrifugation. The precipitate is rinsed by suspending it in 60 ml of buffer A containing 0.1 M magnesium acetate. After 20 minutes, the precipitate is collected by centrifugation and is then extracted for 60 minutes with 80 ml of buffer A containing 0.10 M ammonium sulfate. The supernatant fluid is then collected by centrifugation (fraction III, 76 ml). Step 4. Ammonium Sulfate Fractionation. To 76 ml of fraction I I I (at 0 °) is added 96 ml of saturated ammonium sulfate solution (at 25°). After 15 minutes the precipitate is collected by centrifugation and is ~A trial precipitation is done with aliquots of the solution to determine the minimum amount of protamine sulfate needed to precipitate a maximum amount of enzyme. One milliliter aliquots of fraction II are mixed with from 0.01 to 0.08 ml of 1% protamine sulfate; after 10 minutes the precipitates are removed by centrifugation and the supernatant fluids are assayed. At low concentrations of protamine sulfate (0.01-0.02 ml of 1% protamine sulfate per milliliter of fraction II) a marked enhancement of the enzyme activity in the supernatant liquid is observed. This stimulation may be due to the inactivation or to the selective precipitation of DNase which interferes in the assay. The amount of protamine sulfate to be used in the purification is the amount needed to precipitate 90% of the maximum level of polymerase activity measured in the titration.

[43]

PURIFICATION OF RNA POLYMERASF~

511

then successively extracted with 2.0, 1.8, 1.6, 1.5, and 1.3 M solutions of ammonium sulfate in buffer A. Each extraction is continued for 15 minutes, and the precipitate is then collected by centrifugation for 20 minutes under standard conditions. The first two extractions are carried out with 38-ml aliquots; the last three are carried out with 25-ml aliquots. Fractions containing a high specific activity of RNA polymerase (usually the 1.6 and 1.8M fractions) are pooled, and the enzyme is precipitated by adjusting them to 2.5 M ammonium sulfate with powdered ammonium sulfate. The precipitate is collected by centrifugation and is dissolved in buffer B (fraction IV). Step 5. DEAE-Cellulose Chromatography. A DEAE column is prepared (14 cm length and 2.2 em diameter) and is washed with 500 ml of buffer B. Fraction IV enzyme (30 rag) is diluted with buffer B to a protein concentration of 1 mg/ml and is passed through the column at a flow rate of 1 ml/min. The enzyme is then eluted from the column using a gradient from 0.1 to 0.4 M KC1 in buffer B (total volume 300 ml) with a flow rate of 1 ml/min. The enzyme begins to elute at about 0.16 M KC1. Fractions with a high specific activity are pooled and 0.45 g of ammonium sulfate and 0.004 ml of 1 N NaOH are added together per milliliter of solution with brisk stirring. After the salt dissolves, the solution is stirred gently for 30 minutes. The precipitate is collected by centrifugation and is dissolved in storage solution to give a final protein concentration of about 5 mg/ml (fraction V). For larger amounts of enzyme, the volume of the DEAE column and that of the gradient should be scaled up proportionately. If enzyme of maximum specific activity is desired at this step, one can double the length of the column and the volume of the gradient. This gives a slightly lower recovery of enzyme activity (40-50% overall) but the enzyme obtained is about 90% pure, as estimated by acrylamide gel analysis, and has a specific activity of up to 7400 units/mg. Step 6. Filtration on Biogel P-300. Fraction V enzyme is sufficiently pure for most uses. Step 6 is used when enzyme of maximum purity is required. An aliquot of enzyme fraction V (10-80 mg) is precipitated from storage solution by adding 1.5 volumes of saturated ammonium sulfate. The precipitate is collected by centrifugation and is dissolved in enough buffer B to give a final protein concentration of about 20 mg/ml. The sample is placed on a column of Biogel P-300 (110 cm length and 2.5 cm diameter) which has been equilibrated with buffer B. Elution of the column is carried out by upflow chromatography with buffer B. A constant flow rate of 20 ml/hr is maintained by using an appropriate pump. Fractions which contain a high specific activity of RNA polymerase are pooled, and the enzyme is precipitated and collected as described for fraction V above (fraction ¥ I ) .

512

FACTORS AFFECTING GENE EXPRESSION

[43]

Isolation of Core Polymerase and o] Sigma Component Step 7. Fraction V enzyme (5 mg) is precipitated with 1.5 volumes of saturated ammonium sulfate and the precipitate is dissolved in 0.15 ml of buffer C containing 50 mM KC1. The residual ammonium sulfate is removed by rapid dialysis 1° for 1 hour with three changes of buffer C containing 50 mM KC1 (300 ml each). The dialyzed enzyme is adjusted to a protein concentration of 10-20 mg/ml with the same buffer and is applied to a phosphocellulose column (13.5 cm length and 1 cm diameter) which has previously been washed with at least 20 column volumes of the same buffer. The enzyme is equilibrated with the column for 30 minutes before further elution. The column is then eluted with buffer C containing 50 mM KC1 at a flow rate not greater than 20 ml/hr. Onemilliliter fractions are collected and are immediately monitored for absorbance at 280 m~. An initial peak of contaminating protein appears which contains a low specific activity of sigma. This is followed by a shoulder of absorbance which contains the major fraction of sigma activity. The protein in the shoulder is concentrated on a DEAE column (total volume of 0.5 ml in a Pasteur pipette) which has been equilibrated with buffer C containing 50 mM KC1. This is conveniently carried out by connecting the outlet of the phosphocellulose column directly to the DEAE column after the initial peak of contaminating protein has been eluted. At least 7 column volumes of buffer C containing 50 mM KC1 (50 ml) are washed through the phosphocellulose column onto the DEAE column, since sigma continues to elute from the phosphocellulose column at low concentrations after the initial peak of sigma activity has been eluted. The two columns are then separated, and sigma is eluted from the DEAE column in a small volume of buffer C containing 0.4 M KC1. These fractions are pooled, and the protein is precipitated and stored as described in step 5 (fraction VIIA, sigma component). The core polymerase is eluted from the phosphocellulose column using a 100 ml linear gradient of 0.2 to 0.6 M KC1 in buffer C. The fractions are assayed for core polymerase and holoenzyme using dAT copolymer and T2 DNA templates, respectively. A shoulder of RNA polymerase containing sigma is sometimes found to precede the main peak of core polymerase. Fractions having a high specific activity of core polymerase are pooled, concentrated, and stored as described in step 5 (fraction VIIB, core polymerase). Recoveries of polymerase protein from the phosphocellulose chromatography are low (ca. 50%). This may reflect loss of the protein on the column due to the high ratio of adsorbent to enzyme found necessary ~S. W. Englander and D. Crowe, Anal. Biochem. 12, 579 (1965).

[43]

PURIFICATION OF RNA POLYMERASE

513

to obtain quantitative absorption of the enzyme. Recovery of sigma protein, estimated from the sigma content of the enzyme used for chromatography is very low (ca. 40% overall and 20% in fraction VIIA). The reason for this is not currently understood. Fraction VI enzyme can be used in place of fraction V for step 7. This increases the yield of sigma since step 6 removes low molecular weight contaminants which are otherwise concentrated in the initial peak of the phosphocellulose column.

Reconstitution o] RNA Polymerase Holoenzyme Step 8. RNA polymerase in enzyme fraction V frequently contains less than 1 equivalent of sigma component per mole of core polymerase. RNA polymerase holoenzyme is prepared from this fraction by zone sedimentation in the presence of an added excess of sigma component. The procedure can also be used to reconstitute RNA polymerase holoenzyme from core polymerase. Fraction V enzyme (0.5 mg) is mixed with an aliquot of sigma component (0.035 mg of fraction VIIA) in 1 ml of storage solution. After 10 minutes, 1.5 ml of saturated ammonium sulfate is added, and after 20 minutes the precipitate is collected by centrifugation. The enzyme is dissolved in 0.1 ml of buffer A and is layered onto a linear glycerol gradient in a centrifuge tube (20-40% glycerol in buffer A containing 0.2 M ammonium sulfate). The sample is sedimented for 14 hours at 1° at 50,000 rpm in the SW 50 rotor of the Spinco Model L centrifuge. The tube is punctured and fractions are collected and are analyzed for RNA polymerase and for sigma activity. The fractions containing RNA polymerase are pooled and are concentrated and stored as described in step 5 (fraction VIII). The amount o f sigma needed to saturate core polymerase or fraction V enzyme is determined by titration. Small aliquots (50 ~g) of enzyme are mixed with increasing amounts of sigma component, and each aliquot subjected to zone sedimentation as described above. (The stability of both enzyme and sigma activity in such a trial gradient is improved if the gradient contains 0.4 mg/ml of BSA). Excess sigma component is present when a distinct peak of sigma activity (5 S) is found in the gradient sedimenting more slowly than the polymerase activity (15 S). Alternatively the titration may be followed by analyzing each mixture for free sigma component by eleetrophoresis in acrylamide gels under nondenaturing conditions.11 11j. S. Krakow, K. Daley, and E. Frank, Biochem. Biophys. Res. Commun. 32, 98 (1968).

514

FACTORS AFFECTING GENE EXPRESSION

[43]

TABLE I PURIFICATION OF R N A POLYMERASE

Fraction I. II. III. IV. V.

Extract (NH~)2S04 fraction Protamine eluate (NH4)~S04 reverse extract DEAE-cellulose fraction

Total volume (ml) 183 480 76 24 3.5

Total T2 Specific Recov(units Protein activity ery X 106) (mg/ml) (W2 units/rag) (%) 1.3 0.4 1.7 1.3 1.0

25 5.8 1.2 1.25 4.0

28 13 1.9 X 103 4.3 X 108 6.8 X 103

100 30 132 98 77

Notes on the Purification

Tables I and II show the specific activities and yields during the purification steps described. Assays done on fractions I - I I I have only relative significance since the presence of DNase in the extract and in fractions II and III lowers the amount of incorporation in the assay and hence leads to low values of total enzyme activity. The procedures described for preparation of RNA polymerase and of sigma component are modifications of those previously published.1'm2'13 Steps 1-5 give a good yield of highly purified RNA polymerase which contains sigma component. The procedure can easily be adapted to large-scale preparations (1-10 kg of bacterial cells) by using a highpressure device to disrupt the cells in the initial step. Centrifugations T A B L E II FURTHER PURIFICATION OF R N A POLYMERASE; ISOLATION OF CORE POLYMERASE~ SIGMA COMPONENT~ AND I:~NA POLYMERASE HOLO~NZYME

Fraction V. DEAE-cellulose fraction VI. P-300 fraction a VIIB. Phosphocellulose core polymerase a VIII. Reconstituted a holoenzyme

Total volume (ml)

Total dAT (units X 105)

Specific activity (dAT Protein units/rag Recovery (mg/ml) X 104) (%)

3.5 1.3 1.25

2.8 1.9 8.5

4.0 6.0 4.2

2.0 2.4 1.6

100 66 30

2.0

2.3

5.6

2.0

80

QThe recoveries shown in fractions VI, VIIB, and V I I I are normalized, assuming a constant amount (14 rag) of fraction 5 enzyme as starting material in each step. ~ R. R. Burgess, J. Biol. Chem. 244, 6160 (1969). R. Burgess and A. Travers, this volume [42].

[43]

PURIFICATION OF RNA POLYMERASE

515

during such large-scale preparations are conveniently carried out using a continuous flow centrifuge. Steps 6-8 are carried out on aliquots of fraction V to prepare R N A polymerase holoenzyme, core polymerase, or sigma component as needed. The early fractions in the purification ( I - I V ) are stable enough to allow storage at 0-4 ° overnight. Fractions ¥ through V I I I can be stored at --15 ° in storage solution. Half of the activity is lost from fraction 5 under these conditions after 9 months of storage. These fractions m a y also be stored at -196 ° (liquid nitrogen) in storage solution lacking glycerol. E n z y m e preparations comparable to fraction V have been stored in this manner for several years with no loss in activity. E n z y m a t i c Properties E n z y m e fractions VI and above are essentially free of RNase, 14 DNase, 15 and polynucleotide phosphorylase TM activities, although fraction V m a y contain traces of each. The general requirements for R N A synthesis by R N A polymerase have been previously described, lz Table I I I shows the specific activities of fraction V, core polymerase (fraction TABLE III COMPARISONOF ENZYME AC~VJTmS OBTAINEDWITH dAT AND T~ DNA TEMPLATES Fraction V. DEAE-cellulose fraction VIIB. Core polymerase VIII. Reconstituted holoenzyme

dAT units/mg (X 104)

T~ units/rag

dAT units/ T2 units

2.0 1.6 2.0

6.8 X 103 3 . 0 X 102 7.3 X 103

3.0 53.0 2.8

l~Fraction V enzyme (15 gg) reduced the infectivity of TMV RNA (30 #g) approximately 30% in 30 minutes under standard assay conditions. Enzyme fractions VI-VIII (10 #g) did not cause detectable solubilization (<1%) of 70 pmoles of s2P-labeled X DNA in 60 minutes under assay conditions. Preparations of fraction V did contain DNase detectable by this procedure, causing 4-14% solubilization of the DNA. Fractions VI and VII (1 ~g) did not introduce singlestranded breaks (<20% breakage) in 15 nmoles of intact T7 DNA in 20 minutes. Disappearance of intact single-strands was followed by zone sedimentation of the DNA in alkali. [F. W. Studier, J. Mol. Biol. 11, 373 (1965)]. 1~Measurement of the activity of polynucleotide phosphorylase using the rate of incorporation of 3~P-labeled inorganic phosphate into ADP [Y. Kimhi and U. Z. Littauer, J. Biol. Chem. 243, 231 (1968)], gave specific activities of 0.4-0.6, 0.14, and 1.3 #moles 3~p exchanged per hour per milligram of protein for fractions V, VI, and ¥IIA, respectively. This contaminant was not detectable in fraction VIIB. 17S. Weiss, see Vol XI; J. S. Krakow and S. Ochoa, see Vol VI; S. Ochoa, J. S. Krakow, and C. Basilio, see Vol. VI ; J. Hurwitz, see ¥ol. ¥I.

516

FACTORS AFFECTING GENE EXPRESSION

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VIIB), and RNA polymerase holoenzyme (fraction VIII) obtained using T2 DNA and dAT copolymer as templates. The specific activities of RNA polymerase holoenzyme and of core polymerase are similar when dAT copolymer is used as template, 18 indicating that sigma component is not essential for the initiation or growth of rAU chains. With T2 DNA as template, RNA synthesis is almost totally dependent on the presence of sigma component. With T2 or T7 DNA as template synthesis of specific RNA, that is RNA which is asymmetric or RNA identical to that formed early in phage infection in vivo, is completely dependent on sigma component.19-21 RNA formed by core polymerase with these templates is symmetric and contains RNA species found in both early and late stages of phage infection. The requirement for sigma component for synthesis of T2 RNA can be eliminated if new ends are introduced into the DNA by sonication, or if single-stranded breaks are placed in the DNA by limited treatment with DNase. ~,28 The RNA formed is nonspecific and the activity is apparently due to introduction of nonspecific starting points on the template where the core polymerase can form an active complex. Studies of the attachment of RNA polymerase to T7 DNA have shown that with an intact DNA helix as template, sigma component is not required for the initial attachment of the polymerase to DNA but plays a crucial role in stabilizing this initial complex once attachment has taken place at a specific DNA site.~1 At the present time there is no easily reproducible assay for RNA polymerase which allows characterization of all enzyme fractions with a single template. As a result the characterization of any given enzyme preparation requires enzyme assays done with at least two templates as well as the results of acrylamide gel electropherograms done in the presence of SDS. Poly dAT is not a suitable template for assays during the early stages of purification due to its high sensitivity to E. coli nu~The specific activity of RNA polymerase with dAT template is reduced 12-fold when sigma component is removed. Since sigma constitutes about 20% by weight of RNA polymerase holoenzymea 1.2 fold increase is expected on removal of sigma if both holoenzyme and core polymerase have identical activities with dAT template. Hence the intrinsic activity of core polymerase with dAT template is about 40% lower than that of the holoenzyme. This is confirmed by the finding that when excess sigma component is added to core polymerase there is a 1.8-fold increase in the activity with dAT as template. W. C. Summers and R. B. Siegel, Nature (London) 223, 111 (1969). ~A. A. Travers, Nature (London) 223, 1107 (1969). 21D. Hinkle and M. Chamberlin, Cold Spring Harbor Sympos. Quant. Biol. 35, in press. 22V. Vogt, Nature (London) 223, 854 (1969). :8]:). Hinlde, unpublished observation.

[43]

PURIFICATION OF RNA POLYMERASE

517

cleases. However, dAT is the template of choice during the later stages in purification or with core polymerase since rAU synthesis is affected only to a minor extent by the removal of sigma. Thus only with dAT template can one presently define a specific activity for RNA polymerase which depends primarily on the activity of the enzyme present. A highly fragmented DNA such as calf thymus DNA can also be used as template for core polymerase; however, the exact activities obtained depend critically on the particular DNA preparation used and can vary considerably even during storage of that preparation. The specific activities obtained for RNA polymerase fractions with different dAT preparations appear quite reproducible. While assays with dAT template follow the total polymerase activity during fractionation, assays done with intact T2 DNA are also needed to follow fractionation of the holoenzyme or of sigma component. However, the specific activity of enzyme assayed with T2 DNA is clearly not a good measure of purity of the enzyme since it depends critically on both sigma content of the enzyme and on the intactness of the DNA. Sigma (fraction ¥IIA) has a specific activity of 30,000 sigma units/ mg. It is 90-95% pure as determined by acrylamide gel electrophoresis in SDS buffer. Sigma activity has a half-life similar to that of RNA polymerase holoenzyme when stored at high protein concentrations under the storage conditions described. It is not known whether the specific activity of sigma represents that of essentially homogeneous active sigma, or whether some appreciable fraction of the molecules are inactive. The possibility must be considered that E. coli sigma component consists of a mixture of protein species having the same molecular weight but different in their specificities for individual promoter regions in E. coli DNA.

Physical Properties During the later stages of purification, enzyme fractions are routinely analyzed by acrylamide gel electrophoresis in SDS buffer.~4 This procedure is essential if one is to follow the purity and subunit composition of the polymerase during these steps. Enzyme fraction V contains five major protein components which have been designated as fl', fl, a, a, and ~,1 and about 10% of minor components. The molecular weights of the major components, estimated from their relative migrations during electrophoresis, are 150,000, 145,000, 86,000, 41,000, and 12,000 daltons ___ 10%, respectively.2~ Components fl', fl, and ~ are taken to be subunits ~4A. L. Shapiro, E. Vifiuela, and J. V. Maizel, Jr., Biochem. Biophys. Res. Commun. 28, 815 (1967). u D. Berg, Doctoral Thesis, University of California (1969).

518

FACTORS AFFECTING GENE EXPRESSION

[43]

of the enzyme since they migrate with the enzyme activity through all fractionation procedures and appear in constant ratio to each other. The content of a and ~ m a y v a r y in enzyme fractions depending on the method of preparation. Omega is selectively lost when enzyme is subjected to zone sedimentation in 1 M urea. It is also partially removed when enzyme is analyzed by electrophoresis under nondenaturing conditions. Thus ~ appears to be less tightly bound by the enzyme than are the other components and m a y not be a functional subunit of R N A polymerase. Sigma, which is a functional part of the enzyme, 1,2,2~ can be removed by chromatographing the enzyme on phosphocellulose, by binding it to single-stranded DNA, or by allowing it to engage in RNA synthesis. The relative amounts of each component can be estimated by scanning the stained protein in the gels with a recording spectrophotometer 27 or by scanning negative photographs of the gels with a densitometerY 5 These values, taken with the estimated molecular weights, give approximate molar ratios of the components present, if one assumes that each component stains equally on the basis of mass. 2s B y this criterion fraction VIIB enzyme has a subunit composition of fl,fla~.2~ It contains 0.5% sigma 29 and therefore represents a preparation of RNA polymerase which is 98-99% core polymerase. Fraction V enzyme contains a and the same ratios of fl', fl, ~, and ~ as core polymerase. However, it appears to have only 0.8 equivalent of ~. Thus fraction V enzyme is a mixture of core polymerase and RNA polymerase holoenzyme. If fraction V enzyme is further purified by prolonged zone sedimentation, the resulting enzyme contains only 0.6 equivalents of a. Sigma activity can be found sedimenting more slowly than the enzyme in the gradient. This suggests that a dissociates and can be lost during the course of zone sedimentation. We conclude t h a t the content of a can v a r y appreciably with the method of purification. Consequently, any preparation of R N A polymerase should be analyzed by gel electrophoresis to determine its subunit composition. When R N A polymerase holoenzyme is required, special precautions may be needed to ensure the presence of an equivalent amount of ~. To prepare E. coli B holoenzyme, fraction V enzyme is isolated from a mixture 26R. R. Burgess, J. Biol. Chem. 244, 6168 (1969). ~7A. A. Travers and R. R. Burgess, Nature (London) 222, 537 (1969). 28The validity of this assumption is in question since previous work indicates that proteins can vary by -20% in efficiency of staining by Coomassie blue [S. F. De St. Groth, R. G. Webster, and A. Datyner, Biochim. Biophys. Acta 71, 377 (1963)]. However, the results with fl', fl, and a are consistent with those obtained by Burgess~ using other procedures. ~DA residual amount of sigma is observed in all preparations of fraction VIIB enzyme. This residual sigma is not removed if the preparation is rechromatographed on phosphocellulose.

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PURIFICATION OF RNA POLYMERASE

519

to which an excess of a is added, as described above. Enzyme prepared in this manner contains one equivalent of a and has a subunit composition of fi'flaa.,_~_ as determined by gel electrophoresis in SDS buffer. Core polymerase (fraction VIIB) exists as a single sedimenting species (s~0,w = 12.6 S) at high ionic strength (t~ greater than 0.28). Under these conditions the molecular weight, determined by sedimentation equilibrium, is 3.83 X 1@ +__0.t5 X 10~ daltons, which is consistent with a subunit composition of fl'fl,~2,~2. A molecular weight of 4.0 X 105 ± 0.4 X 105 daltons is estimated for this species from the subunit molecular weight estimates presented above. At low ionic strengths core polymerase aggregates extensively. In the absence of added salt (~ = 0.03) core polymerase sediments as a broad boundary with a mean sedimentation coefficient of 44 ~8S. The width of the sedimenting boundary suggests that the enzyme exists as a mixture of aggregates under these conditions. RNA polymerase holoenzyme (fraction VIII) exists as a single sedimenting species (s2~,~-= 15.0 S) at high ionic strength (t~ greater than 0.15). Molecular weight values for this species, determined by sedimentation equilibrium, have been in the range of 4.0-4.2 X 105 daltons. This is considerably lower than the expected molecular weight of 4.7 }( 105 ± 0.2 X 105 daltons estimated from the molecular weight determined for core polymerase to which 1 mole of sigma component has been added. We attribute the low values obtained by sedimentation equilibrium for the molecular weight of the holoenzyme to a partial dissociation of the holoenzymc to give core polymerase and a during sedimentation. In the absence of added salt (t~ = 0.03) and in the presence of excess a, fraction VIII enzyme sediments at 23 S. Since a dimer of a globular protein having a sedimentation coefficient of 15.0 S has a predicted sedimentation coefficient of 23-24 S, we conclude that RNA polymerase holoenzyme, containing saturating amounts of a, aggregates maximally to give a dimer. Some preparations of fraction VIII enzyme apparently still contain small amounts of core polymerase since they give sedimentation coefficients as high as 25 S at low ionic strength in the absence of additional a.