Journal of Biochemical and Biophysical Methods, 21 (1990) 1-8 Elsevier

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JBBM 00809

Purification of calf thymus RNA polymerase II for in vitro transcription studies J a y a Sivaswami Tyagi * a n d Ira P a s t a n Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, U.S.A.

(Accepted 10 December 1989)

Summary A procedure for the purification of a large amount of RNA polymerase II (RNAP II) from calf thymus is described. This procedure results in an approximately 1400-fold purification with 40% yield of enzyme in 60 h. The partially purified enzyme is highly suitable for in vitro transcription studies in a cell-free system utilizing HeLa S-100. This method for RNAP II purification would find significant applications in in vitro studies for the analysis of factors modulating eukaryotic transcription. Key words: RNA polymerase; Transcription

Introduction Two type of eukaryotic RNAP II-dependent cell-free transcription systems have been developed to investigate the control of gene activity in vitro. The system described by Well et al [1] utilizes purified RNAP II and a crude cytoplasmic extract whereas the Manley system [2] employs a whole-cell extract in the transcription assay. These systems have been utilized to study transcription directly by the promoters of several class II genes [3-6]. On a weight basis, the total amount of RNA polymerase activity in eukaryotic cells is much lower than in prokaryotes. Moreover, difficulties related to enzyme solubilization and instability illustrate the difficulties encountered in studying the regulation of transcription at the molecular level in eukaryotes. RNAP II is an essential component of the Weil system and it is Correspondence address (and * present address): Department of Biotechnology, All India Institute of Medical Sciences, New Delhi-ll0 029, India. 0165-022X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

desirable to purify large amounts of RNAP II rapidly to facilitate in vitro studies of eukaryotic gene activity and to analyse cellular factors that modulate transcription. For tlfis purpose, a simplified procedure for the purification of RNAP H from calf thymus has been developed. This method yields a preparation of the enzyme which is highly suitable for in vitro transcription studies using purified DNA templates. We have used RNAP II purified by this method to isolate and characterize a chick embryo factor tha t inhibits the synthesis of RNAP H-specific transcripts [71.

Materials and Methods

Materials Fresh calf thymus was purchased from a locaI slaughter house. DE52 and P l l were purchased from Whatman. Nucleotides were procured from PL Biochemicals and [3H]UTP and [c~-32p]GTP were from NEN. All other chemicals were purchased from Aldrich, Fisher, BioRad, or Sigma Chemical Co., U.S.A. Buffers BufferA: 10 mM Tris-HC1, pH 7.9, 25 mM KC1, 5 mM MgC12, 0.1 mM EDTA, 12,5% glycerol (v/v) and 0.5% fl-mercaptoethanol. Buffer B: 50 mM Tris-HCl, pH 7.9, 10% glycerol (v/v), 0.1 mM EDTA, 0.5 mM DTT and 0.3 M ammonium sulphate. Buffer C: 50 mM Tris-HC1, pH 7.9, 5% glycerol (v/v), 0.24 g / m l ammonium sulphate 0.1 mM EDTA and 0.5 mM DTT. Buffer D." 50 mM Tris-HC1, pH 7.9, 25% glycerol (v/v), 0.1 mM EDTA and 0.5 mM DTT. Buffer E: Buffer D containing 0.15 M ammonium sulphate. Buffer F: Buffer D containing 0.05 M ammonium sulphate. Buffer G: Buffer D containing 0.05 M ammonium sulphate and 0.2 m g / m l BSA (Miles, Pentex). Buffer H." Buffer D containing 0.22 M ammonium sulphate. Buffer L" Buffer D containing 0.1 M ammonium sulphate. Purification of calf thymus RNAP H All procedures were carried out at 4 ° C unless specified otherwise. A homogenate of 500 g fresh calf thymus was prepared in 1 1 of Buffer A in a Waring blender. The homogenate was clarified by centfifugation at 13 000 × g for 20 rain. PEI precipitation Polyethyleneimine (PEI), to a final concentration of 10%, was added to the supernatant. After stirring for 30 rain, the precipitated proteins were collected by centrifugation as above. The PEI pellet was homogenized in Buffer B and the supernatant containing RNAP II was collected by centfifugation as above.

Ammonium sulphate precipitation Solid ammonium sulphate (0.24 g / m l ) was added to the supernatant and the suspension stirred for 60 rain. A soupy pellet was obtained after centrifugation which was resuspended in Buffer C. A compact pellet was obtained after centrifugation at 34 k rpm for 60 rain in a Beckman ultracentrifuge rotor type 42 and the protein was dissolved in 200 ml of Buffer D and dialyzed against the same buffer for 10 h. DE52 fractionation The concentration of ammonium sulphate was adjusted to 0.15 M and 500 ml of the diluted enzyme was mixed with 500 ml DE52 (settled volume) equilibrated with Buffer E. After 90 min of gentle stirring, the unadsorbed proteins (including RNAP I and III) were filtered off in vacuo. The DE52 was washed extensively with Buffer E and RNAP II was eluted by resuspending it in 500 ml Buffer F for 30 rain. After filtration the DE52 was resuspended in 100 ml Buffer F for 15 rain and refiltered as above. P-11 fractionation The two filtrates were pooled, diluted with Buffer D to a 50 mM ammonium sulphate concentration and then mixed with 65 ml (settled volume) of phosphocellulose (P-11). The enzyme P-11 suspension (4 1) was gently stirred f o r 75 min, filtered and the P-11 was then washed thoroughly with Buffer G. Enzyme bound P-11 was packed into a column (2 × 20 cm) and R N A P II was step-eluted with Buffer H at 50 m l / h . Two-ml fractions were collected in tubes containing 400/~g of BSA and the active fractions were pooled. Enzyme concentration RNAP II was quantitatively precipitated with solid ammonium sulphate (0.55 g/ml). After stirring on ice for 40 rain, the precipitated enzyme protein was collected by ultracentrifugation as above. The AS pellet was resuspended in 1.5 ml of Buffer D and dialyzed vs. the same for 40 min. R N A P II activity was diluted to 300 units/ml prior to storage over liquid nitrogen in 6-/~1 aliquots, flash frozen in dry ice. In vitro transcription assays During enzyme purification, assays were performed in a total volume of 60 /~1 using calf thymus D N A as template. Each reaction contained the following: 3/xmol Tris-HC1, p H 7.9, 36 nmol each of GTP, CTP and ATP, 6 nmol of UTP, 0.5 /~Ci [3H]UTP (sp. act. 25 Ci/mmol), 20 /~g calf thymus D N A and up to 10 /~1 of enzyme. Assays were performed in 100 m M AS and RNAP II activity was determined by its sensitivity to 1 /~g/ml a-amanitin. Samples were processed as described [8]. One unit of enzyme activity represents the incorporation of 1 nanomol of U M P (under saturating conditions) into R N A in 10 min at 37 ° C. In vitro transcription of purified D N A templates were performed as described [1]. Plasmids pSR1 and P C a 2 PRO-3 containing the RSV and the chick collagen a2

(type 1) promoters were used in the assays. Each plasmid was cleaved with the appropriate restriction enzyme and purified prior to use. The following were mixed in a final volume of 50/zl: 25 a M of [a-B2p]GTP (sp. act. approximately 8 C i / m m o l ) , 600/zM each of unlabeled ATP. CTP and UTP, 10 m M Hepes (pH 7.9), 10% glycerol, 60 m M KCI; 7.5 m M MgC1 z, 0.25 m M D T T and 1 /~g appropriate template D N A . Twenty-five #l H e L a S-100 were added per reaction which was initiated by the addition of 0.13 unit of partially purified calf thymus R N A polymerase II. After incubation for 60 rain at 30 ° C. the reactions were terminated b y the addition of 50 #1 of stop solution. R N A s were purified. precipitated and fractionated on 3% polyacrylamide gels and autoradiographed using a published procedure [1].

Protein measurement Protein was measured by the dye-binding assay of Bradford [9] using BSA as a standard.

Results and D i s c u s s i o n

Purification of calf thymus R N A P H Starting from 500 g of calf thymus, 8 mg of partially pure R N A P II was obtained. The procedure takes 60 h to complete and yields 800 units of enzyme of high concentration (700 units/ml, Table 1). The R N A P II preparation contains no more than 3% of R N A P I and III, as measured by activity insensitive to 1 /~g/ml a-amanitin (data not shown). R N A P II has been purified from a variety of eukaryotic sources [10-12]. Published R N A P II purification methods from thymus have employed three to four chromatographic separations followed by glycerol gradient centrifugation [13,14]. In the protocol described here, dialysis times are minimal and column chromatography is avoided until the terminal step elution from P-11. A yield of approximately 40% is achieved b y this method as compared to 8.7% and 11% reported earlier [13,14].

TABLE 1 Purification of RNA polymerase II form calf thymus Purification step

Total activity (units)

Total protein) (mg)

Crude homogenate Ammonium sulphate precipitation DE52 batch elution P-11 step elution

2090 1450

26 648 1432

2130 830

276 a 16 b

a Inclusive of 150 mg BSA; b inclusive of 8 mg BSA.

Specific activity (U/rag thymus protein 0,075 1.01 16.9 103.75

Purification (-fold)

Yield (%)

1 13.5

100 70

225.3 1383.3

101 40

For high recoveries it is essential to start with a large quantity of tissue (0.5-1 kg) and to use batch procedures for ion exchange fractionation. Secondly, pre-cycling phosphocellulose with crude calf thymus proteins increases enzyme recovery. This is achieved by mixing DE52 flow-through proteins with equilibrated P-11 for 4 h and thorough washing of P-11 with Buffer G prior to batch adsorption of R N A P II in the DE52 elution pool. Thirdly, addition of BSA (0.2 m g / m l ) prior to P-11 fractionation increases enzyme stability. Fourthly, high concentrations during storage are essential for enzyme stability. Enzyme concentrations of > 300 units/ml are easily achieved with a terminal ammonium sulphate precipitation. The R N A P II prepared as described here has remained stable for at least 30 months over liquid nitrogen. The three largest subunits of calf thymus RNAP II, of 215. 175 and 140 kDa, as described in the literature [8], are visible in the SDS-PAGE profile (Fig. 1. lanes 3 and 4) suggesting that the RNAP II preparation is a mixture of forms IIA and liB. For in vitro transcription studies it is not necessary to purify the enzyme to homogeneity. It is, however, essential that the preparation be free from contaminating nucleases and phosphatases and that it be totally dependent on exogenously-ad-

Fig. 1. SDS-polyacrylamide gel eleetrophoresis of calf thymus R N A polymerase II. R N A P II subunits were fractionated on a 7% SDS-polyacrylamide gel. Electrophoresis was performed at 17 m A for 8 h and protein bands were stained with Coomassie brilliant blue. Lane (1), molecular mass markers: phosphorylase b 98 kDa, BSA 67 kDa, ovalbumin 48 kDa; lane (2), E. coli R N A polymerase, fl' subunit 160 kDa, fl subunit 155 kDa, o subunit 86 kDa, a subunit 40 kDa; lanes (3 and 4), 2 and 10/zg P-11 pool of calf thymus R N A P II Prep II; lane (5) 10/xg Sephacryl S-200 pool of calf thymus R N A P II.

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Fig. 2. Analysis of RNAP II specific RNA synthesis from the a2 (type I) collagen promoter and the RSV promoter. RNAs were synthesized in vitro as described in Materials and Methods. In panel A the template used was the chick a2 (Type I) coltagen promoter containing plasmAd pCa2PRO-3 linearized with BamHI, in panel B the template was HinclI-cleaved pSR1 contahiLng the RSV promoter. RNAs were synthesized for 0, 5, 10, 15, 30, 60 and 120 rain for lanes 1-7, respective!y. The specific transcripts indicated by the arrow, were 450 bases long in A and 440 bases long in B. The a-amanifin-sensifive transcript from each lane was excised from the gel and counted by Cerenkov counting. A counting efficiency of 20% was accounted /or and the results obtained are plotted in panel C. Collagen-specific transcript (~, higher scale), RSV-specific transcript (o, lower scale).

ded DNA. The enzyme purified up to the P-11 step by the method described here satisfies these two requirements. However. several contaminant proteins are seen in the R N A P II preparations purified up to the P-11 step. Contaminant proteins smaller than 68 kDa are effectively removed by Sephacryl S-200 chromatography (column 1.5 cm × 85 cm) using Buffer I and a 4150-fold purification is achieved (Fig. 1. lane 5). This is similar to a 4500-fold purification reported earlier [14]. However. when equal amounts of RNAP II (0.13 units) of the S-200 pool and the P-11 pool are added in the transcription assay, equal incorporation of [a-32p]GTP into promoter-specific transcripts is observed (data not shown). This indicates that Sephacryl S-200 chromatography did not remove any contaminants adversely affecting R N A P II-specific transcription in the cell-free system. Therefore. enzyme purified up to the P-11 step is adequate for in vitro transcription assays.

In vitro transcription of truncated DNA templates The suitability of the partially purified R N A P II in a cell-free transcription system was evaluated using two recombinant plasmids pCa2PRO-3 and pSR1 containing the collagen a2 and the RSV promoters, respectively. These plasmids were linearized with BamHI and HincII, respectively, and added separately to the in vitro transcription assay as described [7]. Incubations were performed with enzyme purified up to the P-11 step in the presence and absence of e~-amanitin to differentiate RNAP II-catalyzed transcription from other R N A synthesis. The electrophoretic mobility of the a-amanitin-sensitive R N A was compared with the corresponding transcript in the Manley system. Incubation of BamHI-cleaved pC~2PRO-3 in the cell-free system yields runoff transcripts approximately 450 bases in length (Fig. 2A). A 440-bases-long transcript is obtained with pSR1 cleaved with HincII (Fig. 2B).The transcript sizes obtained are similar to those obtained in the Manley system using these promoters [15,16]. In both instances, R N A synthesis is D N A dependent, actinomycin D sensitive and completely blocked by ~-amanitin at 1 /xg/ml, confirming R N A P II catalyzed transcription. Two different eukaryotic promoters directed the synthesis of transcripts similar in length to those reported earlier, thus establishing that the calf thymus RNAP II promotes accurate initiation of transcription in this system. The stability of RNAP II in the in vitro transcription system was also evaluated. RNAP II remains active up to at least 2 h at 30 ° C, as shown in Fig. 2C. At 15 min the intensity of the RSV and collagen-specific transcripts are approximately equal, though the RSV transcript appears to be degraded from 1 h ~onwards (Fig~ 2B). That this is not due to nuclease contamination is clear from the steady accumulation of the collagen transcript up to 2 h (Fig. 2A and C).

Simplified description of the method and its application A simplifiedprocedure for the purification of RNA polymeraseII from calf thymus is described. An approximatelyM00-foldpurification and nearly 40% yield of enzymeis achieved.The procedure includes

a PEI precipitation, ammonium sulphate precipitation, DE52 batch chromatography and step elutioa from a batch-absorbed P-11 column. The enzyme thas purified is Nghly suitable for in vitro transcription studies using purified DNA templates.

Acknowledgements W e t h a n k T. Y a r n a m o t o

and P.A. Weil for useful discussions.

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