Proc. Natl. Acad. Sci. USA Vol. 94, pp. 11869–11874, October 1997 Biochemistry

Extensive purification of a putative RNA I holoenzyme from plants that accurately initiates rRNA gene in vitro (nucleolus͞rDNA͞gene expression)

JULIO SAEZ-VASQUEZ AND CRAIG S. PIKAARD*

Biology Department, Washington University, Campus Box 1137, One Brookings Drive, St. Louis, MO 63130

Edited by Masayasu Nomura, University of California, Irvine, CA, and approved August 25, 1997 (received for review May 19, 1997)

ABSTRACT RNA polymerase I (pol I) is a nuclear Encouraged by brief reports that -free rRNA gene whose function is to transcribe the duplicated genes encoding transcription could be achieved in plant extracts (22, 23), we set the precursor of the three largest ribosomal RNAs. We report out to develop a cell-free system from broccoli (Brassica a cell-free system from broccoli (Brassica oleracea) inflores- oleracea) to further our studies of rRNA gene regulation in cence that supports -dependent RNA pol I transcrip- Brassica and Arabidopsis (24–28). We show that a pol I- tion in vitro. The transcription system was purified extensively containing activity sufficient for promoter-dependent tran- by DEAE-Sepharose, Biorex 70, Sephacryl S300, and Mono Q scription can be purified extensively, with biochemical prop- chromatography. Activities required for pre-rRNA transcrip- erties suggesting a single complex. Approximately 30 polypep- tion copurified with the polymerase on all four columns, tides are present in fractions purified to near-homogeneity, suggesting their association as a complex. Purified fractions consistent with the estimated mass of the putative holoenzyme programmed transcription initiation from the in vivo start site complex and the expected complexity of the pol I transcription and utilized the same core promoter sequences required in system. vivo. The complex was not dissociated in 800 mM KCl and had a molecular mass of nearly 2 MDa based on gel filtration MATERIALS AND METHODS chromatography. The most highly purified fractions contain Ϸ30 polypeptides, two of which were identified immunologi- Preparation of Broccoli Nuclear Extracts. Nuclear extracts cally as RNA polymerase subunits. These data suggest that the were prepared from chilled (4°C) broccoli inflorescence pur- occurrence of a holoenzyme complex is probably not unique to chased at a local supermarket, with all steps performed at 4°C. the pol II system but may be a general feature of eukaryotic After removing stalks and large stems, each 100–125 g of nuclear . inflorescence was added to 200 ml of ice-cold homogenization buffer (29) [0.44 M sucrose͞1.25% Ficoll͞2.5% Dextran͞20 Eukaryotic nuclear RNA polymerases interact with transcrip- mM Hepes, pH 7.4͞10 mM MgCl2͞0.5% Triton X-100 sup- tion factors to recognize gene promoters and initiate tran- plemented just prior to use with 0.5 mM DTT͞5.0 ␮g/ml scription. Many of the required factors can be purified indi- antipain͞3.5 ␮g/ml bestatin͞5.0 ␮g/ml leupeptin͞4.0 ␮g/ml vidually and added back together to reconstitute transcription pepstatin A͞0.5 mM phenylmethylsulfonyl fluoride (PMSF)] in vitro, allowing their identification and molecular character- and homogenized at maximum speed in a Waring blender ization (1–7). However, proteins not required under a given set using five pulses of 20 sec each. The homogenate was filtered of assay conditions can be overlooked, though they may play through two layers of Miracloth (Calbiochem) and nuclei were important roles in vivo. The latter insight has come, in part, pelleted at 9,400 ϫ g for 30 min. The pellet was resuspended from the characterization of RNA pol II holoenzyme com- gently in 50 ml of resuspension buffer (RB; 50 mM Hepes, pH plexes in yeast and mammals (8–12). These complexes are 7.9͞20% glycerol͞10 mM EGTA͞10 mM MgSO4.7H2O͞0.5 several megadaltons in size and include pol II core enzyme mM PMSF͞0.5 mM DTT), and then solid NaCl was added to subunits, most of the general transcription factors, and a a final concentration of 1.0 M. The sample was mixed on a growing list of additional activities including protein stirring motor for Ϸ10 min, then 1.0 ml of 50% (wt͞vol) (13), chromatin remodeling activities(14), and proteins in- polyethylene glycol (PEG 8000) was added, and stirring con- volved in DNA repair (12). tinued 20 min. Following centrifugation at 17,000 ϫ g for 30 There has been at least one report of a pol I-containing min., the supernatant was diluted 8-fold with RB buffer. fraction from rat cell-free extracts sufficient for accurate Proteins were precipitated with solid (NH4)2SO4 added to 0.33 transcription in vitro (15), and several studies have shown that g͞ml over a 20-min period, followed by incubation at 4°C for pol I transcription factors interact. For instance, the vertebrate 1 hr and centrifugation at 17,000 ϫ g for 30 min. Precipitated proteins were resuspended in RB containing 100 mM KCl pol I transcription factors UBF and SL1͞TIF-IB interact both in solution and on the DNA (16–18). Likewise, UBF and pol (RB100) to a final volume of 12 ml per 100–125 g of starting I cosediment in glycerol gradients and interact on Far-Western tissue. Typical protein concentrations were Ϸ1.5–2.0 mg͞ml as blots (19). Two additional factors, TIF-IA and TIF-IC copurify estimated by the method of Bradford (30) using BSA as the with pol I on multiple columns (20, 21). Collectively, these standard. Assay for Nonspecific RNA Polymerase Activity. results suggest that the pol I transcription factors might be Promoter- associated with one another, possibly within a complex anal- independent (nonspecific) polymerase activity was measured ogous to the pol II holoenzyme. using standard assays (31–33). Crude extract or column frac- tion (20 ␮l) was mixed with 20 ␮lof2ϫreaction mix containing

The publication costs of this article were defrayed in part by page charge This paper was submitted directly (Track II) to the Proceedings office. payment. This article must therefore be hereby marked ‘‘advertisement’’ in Abbreviations: pol I, II, and III, RNA polymerase I, II, and III; FPLC, accordance with 18 U.S.C. §1734 solely to indicate this fact. fast protein liquid chromatography. © 1997 by The National Academy of Sciences 0027-8424͞97͞9411869-6$2.00͞0 *To whom reprint requests should be addressed. e-mail: pikaard@ PNAS is available online at http:͞͞www.pnas.org. biodec.wustl.edu.

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100 mM Hepes-KOH (pH 7.9), 100 mM KCl, 4 mM MnCl2,1 electrophoresis on 6% polyacrylamide͞7 M urea sequencing mM each rATP, UTP, rCTP, 0.08 mM unlabeled rGTP, 2 gels (34). Digestion products were visualized by autoradiog- mg͞ml BSA, 50 ␮g͞ml sheared calf thymus DNA, 100 ␮g͞ml raphy or by a PhosphorImager (Molecular Dynamics). ␣-amanitin (Sigma), and 10 ␮Ci (1 Ci ϭ 37 GBq) ␣-labeled [32P]rGTP ([32P]UTP was used initially with similar results). After 30 min at 30°C, reactions were spotted on DEAE RESULTS (Whatman DE81) filters. Unincorporated label was removed Development of a Plant in Vitro Transcription System for with four washes in 0.5 M sodium phosphate (5 min each) RNA Pol I. Broccoli inflorescence (the part we generally eat) followed by washes in 95% ethanol (twice) and diethyl ether to is an inexpensive source of rapidly dividing cells in which pol aid drying. Incorporated radioactivity was determined by I transcription is expected to be very active. Soluble proteins scintillation counting. Background was determined from mock extracted from crude nuclei were precipitated, dialyzed into reactions lacking added protein. 100 mM KCl buffer (RB100), and purified first by step Purification of RNA Pol I. Nuclear extracts were purified fractionation on a DEAE-Sepharose anion-exchange column using three or four chromatography steps (see Figs. 1A and (Fig. 1A). Approximately 15% of total ␣-amanitin-resistant 4A). The first two steps were the same in both schemes. For the data of Figs. 1–3, Ϸ50 ml of ammonium sulfate- nonspecific polymerase activity (assayed on nicked calf thymus concentrated nuclear extract was loaded onto a 20 ml DEAE DNA) flowed through the DEAE column in RB100. An Sepharose CL-6B (Pharmacia) column equilibrated in RB100. The flow-through was collected and the column washed with RB100. Bound proteins were eluted with successive RB200 and RB400 steps. The 400 mM KCl-eluted fraction (DEAE 400) was dialyzed against RB100 then loaded ontoa2ml Biorex 70 (Bio-Rad) column equilibrated in RB100. The column was washed with RB100 and eluted with RB800. This Biorex 800 fraction was dialyzed against column buffer (CB; 25 mM Hepes, pH 7.9͞20% glycerol͞0.1 mM EDTA͞0.5 mM DTT) containing 100 mM KCl (CB100) and then subjected to chromatography ona2mlanalytical Mono Q fast protein liquid chromatography (FPLC) column (Pharmacia). The Mono Q column was washed with CB100 then eluted with a 20 ml linear gradient from CB100 to CB600. Fractions (1 ml) were dialyzed against RB100 then assayed for nonspecific and promoter-dependent transcription. In the four-column purification scheme, the Biorex 800 fraction (in Ϸ3 ml) was loaded directly onto a 195 ml Sephacryl S-300 (Pharmacia) FPLC column (Ϸ97 cm long) equilibrated and run in CB100. Peak polymerase-containing fractions were subjected to Mono Q chromatography as described above. DNA Templates and in Vitro Transcription Reactions. Plas- mid pBor2 contains B. oleracea rRNA gene promoter se- quences from Ϫ518 to ϩ106 (26). The Arabidopsis thaliana rRNA gene promoter clone pAt1 contains sequences from Ϫ520 to ϩ92. pAt1 and its deletion derivatives have been described (24, 25). For in vitro transcription reactions, 20 ␮lof Mono Q purified protein was mixed with 125–1,000 ng (see figure legends) of supercoiled pBor2 or pAt1 plasmid DNA. Following a 5-min incubation at room temperature, 20 ␮lof2ϫ reaction mixture (30 mM Hepes, pH 7.9͞80 mM potassium FIG. 1. Purification of broccoli (B. oleracea) fractions that program acetate͞12 mM magnesium acetate͞1mMDTT͞200 ␮g/ml accurate transcription initiation in vitro.(A) Purification scheme and ␣-amanitin͞1 mM of each nucleotide triphosphate) was added. summary data. Approximately 70 mg of nuclear protein was fraction- Transcription reactions were incubated2hat25°C and ated by step elution on DEAE-Sepharose and Biorex 70, followed by terminated by addition of 360 ␮l of stop solution [150 mM linear gradient elution from Mono Q using FPLC. Protein concen- NaCl͞50 mM Tris⅐HCl, pH 8.0͞250 mM sodium acetate, pH tration and nonspecific polymerase activity in the presence of 100 5.3͞3 ␮g/ml yeast tRNA͞6 mM EDTA, pH 8.0] followed by ␮g͞ml ␣-amanitin was measured for each fraction. One unit of pol extraction with an equal volume of phenol͞chloroform and activity is defined as 1,000 cpm incorporated in the nonspecific assay vortex mixing. Following brief centrifugation in a microcen- (see Materials and Methods). (B) Elution profile of total protein and 4 nonspecific polymerase activity on Mono Q using the fractionation trifuge, the aqueous phase was mixed with Ϸ2 ϫ 10 cpm of scheme depicted in A. The column was eluted with a 10-column probe DNA (see figures) 5Ј end-labeled with T4 polynucle- volume linear gradient from 0.1 M to 0.6 M KCl. Eluted proteins were 32 otide and ␥-labeled [ P]ATP (34). RNA transcripts monitored continuously by UV absorbance. Fractions 10–20 were and probe were coprecipitated with ethanol and resuspended assayed for nonspecific polymerase activity in the presence of 100 in 30 ␮l of hybridization buffer (80% formamide͞40 mM Pipes, ␮g͞ml ␣-amanitin (dashed line). (C) Single Mono Q fractions pro- pH 6.4͞400 mM NaCl͞1 mM EDTA). After initial denatur- gram accurate transcription initiation from the B. oleracea rRNA gene ation for 10 min at 100°C, hybridization was carried out at 37°C promoter. Fractions 9–19 were mixed with 500 ng of pBor2 in reactions overnight. A total of 270 ␮l of S1 digestion buffer (34) containing 100 ␮g͞ml ␣-amanitin. Transcripts were detected by S1 containing 125 units͞ml S1 nuclease (Sigma) was added. S1 nuclease protection using a probe that spanned the transcription start site (see diagram). The labeled XbaI site is derived from the plasmid digestion reactions were incubated 30 min at 37°C and stopped adjacent to the cloned promoter sequences thus the probe only detects by addition of 5 ␮l 0.5 M EDTA. Following addition of 30 ␮l transcripts derived from the plasmid. Dideoxynucleotide sequencing 7.5 M ammonium acetate, reaction products were ethanol reactions, using a primer end-labeled at the same nucleotide as the S1 precipitated. Following centrifugation, pellets were resus- probe, were run in adjacent lanes to verify initiation at the in vivo start pended in 95% formamide loading dye and subjected to site TATATAAGGG (ϩ1 is underlined). Downloaded by guest on September 26, 2021 Biochemistry: Saez-Vasquez and Pikaard Proc. Natl. Acad. Sci. USA 94 (1997) 11871

additional 35% was eluted with RB200, followed by a final In vivo, the upstream boundary of the A. thaliana rRNA gene Ϸ50% that eluted with RB400 (DEAE 400 fraction). RNA pol promoter was shown to be between Ϫ55 and Ϫ33 and is III in Brassica is highly resistant to ␣-amanitin and is inhibited probably closest to Ϫ33 (25). This conclusion is based on four Ͻ50% even at 1 mg͞ml (33). Thus the contributions of pol I transient expression experiments in which a Ϫ55 (pAt1 5Ј⌬-55) and pol III to the activity detected in DEAE fractions is not deletion construct was always functional. In two trials, weak discriminated in the nonspecific assay. However, the DEAE expression was detected from a promoter deleted to Ϫ33. 400 fraction, which accounted for 13% of the starting aman- However, in the other two experiments, transcripts from the itin-resistant polymerase activity, programmed weak transcrip- 5Ј⌬-33 construct were not detected. The downstream pro- tion from the in vivo start site using the B. oleracea rRNA gene moter boundary was mapped by transient expression using promoter construct pBor2 (data not shown). DEAE 100 and constructs whose 5Ј boundary was Ϫ520, but whose 3Ј se- DEAE 200 fractions lacked comparable activity. Combining quences were sequentially deleted (25). Deletion to ϩ6 had no DEAE fractions did not further stimulate nonspecific or effect on promoter activity, but further deletion to positions promoter-dependent transcription. upstream of the transcription start site abolished transcription. We used these same 5Ј and 3Ј deletion constructs to test the Following dialysis against RB100, the DEAE 400 fraction promoter sequences required in vitro with the peak Mono Q was loaded onto a Biorex 70 cation-exchange column equili- fraction purified as in Fig. 1. Deletion of 5Ј sequences from brated in RB100. Greater than 90% of the nonspecific poly- Ϫ520 to Ϫ380, Ϫ110, Ϫ55, or Ϫ33 all yielded similar levels of merase activity was retained on the column and was eluted in accurately initiated transcripts in vitro (Fig. 3A, lanes 1–5). a single step with RB800; the remaining Ϸ10% flowed through Further deletion to Ϫ6 abolished transcription (lane 6), indi- the column. The yield of polymerase activity in the Biorex 800 cating that the 5Ј promoter boundary in vitro is between Ϫ33 fraction was Ϸ3% of the starting activity (Fig. 1A). and Ϫ6. Likewise, 3Ј deletions to ϩ47, ϩ18, or ϩ6 were fully Following dialysis against CB100, the Biorex 800 fraction functional for transcription (Fig. 3B; lanes 1–3, respectively), was subjected to chromatography on an analytical Mono Q but further deletion to Ϫ13 abolished transcription (lane 4). anion exchange column and eluted with a 20 ml linear salt Read-through transcripts detected in Fig. A (see lanes 4 and 5 gradient from CB100 to CB600 (Fig. 1B). Individual fractions in the region denoted by an asterisk) and B are due to pol I were tested for nonspecific pol I activity and the ability to transcripts that traverse the promoter region from upstream program promoter-dependent pol I transcription. Nonspecific due to the use of circular plasmid templates. pol I activity eluted at Ϸ450 mM KCl and peaked in fractions The results of Fig. 3 agree closely with those obtained in vivo. 14–16. These same fractions initiated transcripts from the B. The major difference was that the 5Ј⌬-33 construct was oleracea rRNA gene promoter, with fraction 16 representing expressed at approximately the same level as the Ϫ55 construct the peak of promoter-dependent activity (Fig. 1C). Transcrip- in vitro whereas in vivo, the Ϫ33 construct was only weakly tion signals remained fairly uniform using a broad range of expressed, at best. A parallel is that in mammalian systems, template concentrations and did not display template inhibi- core promoter sequences are often sufficient to program tion at any concentration tested (Fig. 2 and data not shown). transcription in vitro, but additional upstream sequences are The Brassica Pol I-Containing Protein Complex Transcribes needed for efficient transcription in vivo (35–38). the Arabidopsis Promoter and Requires the Same Promoter Purification of the Putative Pol I Holoenzyme to Near- Sequences Needed in Vivo. The B. oleracea promoter is active Homogeneity. The experiments above suggested that the Mono when transfected into cells of A. thaliana, a related species (26). Q fractions contain all activities required for core promoter Therefore, we expected an A. thaliana rRNA gene promoter to recognition and transcription initiation. If these activities were be transcribed in the broccoli in vitro system, allowing pro- associated within a single complex, we reasoned that they moter deletion mutants already characterized by transient should also copurify when fractionated by size. Therefore, we subjected an undialyzed Ϸ3 ml Biorex 800 fraction to gel expression (24, 25) to be used to compare promoter require- filtration chromatography using a 195 ml Sephacryl-S300 ments in vitro and in vivo. As predicted, transcripts were column equilibrated and run in CB100 (Fig. 4A). Nonspecific accurately initiated from the Arabidopsis promoter construct pol I activity, and a significant portion of the total protein, pAt1 (Fig. 3A, lane 1). The Brassica promoter was only slightly eluted as a sharp peak followed by a trailing shoulder of activity stronger than the pAt1 promoter construct in side-by-side (Fig. 4B). The major peak eluted with the trailing edge of Blue reactions (less than 2-fold; data not shown). Dextran, which has an average molecular weight of 2 MDa. A standard curve generated by plotting relative elution volume versus the log of the molecular mass for Blue Dextran (2 MDa assumed as the mass of the peak), thyroglobulin (669 kDa), ferritin (450 kDa), and BSA led to an estimated mass of Ϸ1.7 MDa ϮϷ0.3 MDa) for the pol I-containing peak. The trailing shoulder of pol I activity tailed off at Ϸ500 kDa, the approx- imate size of the pol I core enzyme (39). Peak pol I fractions from the Sephacryl column were pooled and subjected to chromatography on Mono Q. Elution of the Mono Q column as in Fig. 1 led to a single major UV-absorbing peak eluting at Ϸ450 mM KCl (Fig. 4C). The peak of non- specific pol I activity corresponded to precisely these same fractions (Fig. 4C), as did promoter-dependent pol I transcrip- tion activity (Fig. 4D). The fact that the pol I activity eluted from Mono Q with the FIG. 2. Accurate transcription initiation programmed by peak only major protein peak suggested that the complex was Mono Q fractions is relatively insensitive to template concentration. purified to near-homogeneity. Therefore peak fractions Totals of 125, 250, 500, or 1,000 ng (lanes 2–5, respectively) of pBor2 plasmid DNA was added to transcription reactions containing the peak throughout the purification scheme (see Fig. 4A) were com- Mono Q fraction (no. 16) in the presence of 100 ␮g͞ml ␣-amanitin. In pared qualitatively (Fig. 5A). The polypeptide composition of lane 1, plasmid DNA not containing an rRNA gene promoter was the last two peak fractions (Sephacryl and Mono Q) was very present in an equivalent reaction. A dideoxy sequencing ladder was similar, suggesting that the complex was approaching purity (at generated and run beside the reactions as in Fig. 1. which point no further purification is achieved by subsequent Downloaded by guest on September 26, 2021 11872 Biochemistry: Saez-Vasquez and Pikaard Proc. Natl. Acad. Sci. USA 94 (1997)

FIG. 3. rRNA gene promoter sequences between Ϫ33 and ϩ6 are required for transcription in vitro. A series of 5Ј (A) and 3Ј (B) A. thaliana promoter deletion constructs derived from pAt1 were tested in vitro using the peak Mono Q fraction (purified according to Fig. 1) in the presence of 100 ␮g͞ml ␣-amanitin. A total of 500 ng of each plasmid was tested, and transcripts were detected by S1 protection using the probes shown at the bottom of the figures. Different probes had to be used for each 3Ј deletion construct; thus the protected fragments are different sizes. Reactions labeled ‘‘mock’’ were performed using Mono Q fractionated protein but no template.

steps). Approximately 30 distinct protein bands are apparent DEAE, Biorex, gel filtration, and Mono Q chromatography in the final peak Mono Q fraction, resolved best on a 4–20% yielded a single major protein peak on the final column, an gradient SDS͞polyacrylamide gel (Fig. 5B). These include indication of purity or near-purity. Approximately 30 distinct proteins of the appropriate size to be subunits of pol I core polypeptides are present in such peak fractions, two of which enzyme, whose expected positions are denoted by asterisks in cross-react with the only two currently available antibodies Fig. 5. Two of these, migrating at Ϸ15 kDa and 27 kDa, were against proteins expected to be plant pol I subunits. The sum confirmed as polymerase subunits by Western blot analysis of the masses of the Ϸ30 protein bands is Ϸ2 MDa, in using antibodies generously provided by Robert Larkin and reasonable agreement with the Ϸ1.7 MDa estimate from Tom Guilfoyle (University of Missouri). Antisera against the Sephacryl chromatography. Thirty proteins are also a plausible 14.1 kDa (predicted) Arabidopsis homolog of the yeast pol complexity for the pol I transcription system. In mouse, pol I I͞pol III subunit AC19 (40) cross-reacted with a protein of the core enzyme (11 subunits) (41), SL1͞TIF-IB (4 subunits), appropriate size in Mono Q fractions (Fig. 5C) purified UBF, TIF-IA, and TIF-IC account for at least 18 proteins. In according to the three column procedure diagrammed in Fig. yeast, 24 known proteins participate in pol I transcription: the 1A (lanes 1 and 2) or the four column procedure of Fig. 4 (lane pol I core enzyme is composed of 14 subunits (42), a complex 3). The Western blot analysis result with the anti-27-kDa of 4 proteins recognizes the core promoter (TBP, Rrn proteins subunit antibody is not shown because Larkin and Guilfoyle 6, 7, and 11) (43, 44), a complex of 5 proteins interacts with the have not yet published the cloning and characterization of this upstream promoter domain (Rrn proteins 5, 9 and 10 and two subunit. unidentified proteins) (45), and Rrn3p interacts with the Though silver-staining intensity is often variable between polymerase core enzyme in solution (46). proteins, the major bands apparent in Fig. 5B appear to be The fact that the DEAE 400 fraction programmed accurate comparable in abundance to the small polymerase subunits transcription from the Brassica promoter was not unexpected verified by Western blot analysis. Minor bands may be break- because a similar DEAE fraction programs in vitro pol I down products of larger proteins, proteins associated with transcription in yeast, Xenopus, and mammals (15, 47, 48). polymerase in substoichiometric amounts, proteins that do not Subsequent copurification of all necessary factors on Biorex, stain well with silver, or contaminating proteins that copurify Sephacryl and Mono Q was more surprising, but is also true in with RNA pol I on all four columns. Xenopus (A.-C. Albert, M. Denton, and C.S.P., unpublished work). Glycerol gradient sedimentation of crude or partially DISCUSSION purified Xenopus cell extracts also results in single fractions capable of promoter-dependent transcription (M. Denton and We have developed a cell-free transcription system from C.S.P., unpublished work). It seems unlikely that all essential broccoli (B. oleracea) that is ␣-amanitin insensitive, initiates transcription factors copurify with polymerase by virtue of transcription from the proper start site, and requires the same having the same elution characteristics on multiple anion promoter regions needed in vivo. These initial results sug- exchange, cation exchange, and gel filtration columns. Instead, gested that our fractions contained an activity that could the simplest explanation is that these activities are associated, account for the known attributes of pol I transcription in vivo. comprising a holoenzyme complex. We point out that a protein Subsequent purification of transcriptionally active fractions by complex, known as a , can be separated from the pol Downloaded by guest on September 26, 2021 Biochemistry: Saez-Vasquez and Pikaard Proc. Natl. Acad. Sci. USA 94 (1997) 11873

FIG. 4. Purification of the putative B. oleracea pol I holoenzyme to near homogeneity. (A) Fractionation scheme and summary data. Following DEAE and Biorex chromatography, the Biorex 800 fraction was loaded directly onto a 195 ml Sephacryl S300 column equilibrated and run in 100 mM KCl buffer (see B). Peak pol I-containing fractions FIG. 5. Polypeptide composition of highly purified holoenzyme were then subjected to Mono Q chromatography (see C). (B) Elution fractions. (A) Equal amounts of protein from crude nuclear extract, profile of total protein and nonspecific pol I activity on Sephacryl S300. the DEAE 400 mM KCl fraction, Sephacryl S300 peak, and Mono Q Proteins were detected by UV absorbance at 280 nm. Individual peak fractions were subjected to electrophoresis on an 8.5% SDS͞ fractions were then tested for ␣-amanitin insensitive nonspecific polyacrylamide gel and visualized by silver staining. (B) The Mono Q transcription. Peak elution positions for Blue Dextran, thyroglobulin, peak fraction was also subjected to electrophoresis on a 4–20% ferritin, and BSA molecular mass standards are indicated. (C) Mono gradient SDS gel. Asterisks indicate the expected sizes for subunits of Q chromatography of Sephacryl peak fractions was performed as in the pol I core enzyme based on the work of Guilfoyle (39). Arrows Fig. 1. Fractions were dialyzed and tested for ␣-amanitin-resistant labeled Ab indicate polymerase subunits verified by Western blot polymerase activity. The major UV-absorbing protein peak corre- analysis (C and data not shown). (C) Western blot analysis using an sponded to the peak of polymerase activity. (D) Peak Mono Q antibody against the Arabidopsis homolog of the yeast AC19 polymer- fractions program accurate transcription initiation. Aliquots of the ase subunit. Lanes: 1 and 2, Mono Q peak fraction (two amounts) Mono Q peak were added to transcription reactions containing 0, 250, following DEAE and Biorex (D-B-Q fractions; see Fig. 1A); 3, Mono or 500 ng of pBor2 plasmid DNA in the presence of 100 ␮g͞ml Q peak fraction after the four column scheme of Fig. 4A (D-B-S-Q; ␣-amanitin. Transcripts were detected by S1 nuclease protection. lane 3); 4, 30 ng of the purified recombinant 14.1-kDa (predicted size) protein used to raise the antibodies (gift of R. Larkin and T. Guilfoyle). II holoenzyme under certain conditions (49); thus the ability Enhanced chemiluminescence detection was used in lanes 1 and 2, and to fractionate a transcription system into multiple activities colorimetric detection was used in lanes 3 and 4. Proteins were does not show definitively that the activities do not associate resolved on a 12.5% SDS͞PAGE gel prior to blotting to nitrocellulose. as metastable complexes in the cell. Pre-immune serum did not cross-react on duplicate blots (data not Polymerase activity in different extracts of store-bought shown). broccoli is variable (see Figs. 1A and 4A) but does not affect the purification scheme. More difficult to interpret is the yield to form holoenzyme complexes, in reasonable agreement with of pol I activity and what this implies about the abundance of our data. the putative holoenzyme. Yields of 1–2% (Figs. 1A and 4A) Without knowing the exact ratio of free polymerase to are slight underestimates given that both pol I and pol III holoenzyme, it is difficult to estimate the degree of purification contribute to amanitin-resistant transcription in starting ex- we have achieved. Based on initial ␣-amanitin resistant poly- tracts, but such yields are not unreasonable. In human cells, it merase activity, purification of the holoenzyme to near- is estimated that there are several hundred copies of SL1, but homogeneity results in only a 26-fold increase in polymerase 104–105 pol I molecules (16). Likewise in yeast, core factor and specific activity (Fig. 4A). However, if only 5–10% of the upstream activation factor complexes appear to be present in starting polymerase activity were pol I holoenzyme (and hundreds of copies per cell whereas polymerase is estimated at 90–95% were pol III and pol I core enzyme), the degree of Ϸ104 copies (45). These estimates suggests that no more than holoenzyme purification would be 260- to 520-fold. We also 2–10% of pol I could be associated with transcription factors expect Ϸ3- to 4-fold lower polymerase activity per microgram Downloaded by guest on September 26, 2021 11874 Biochemistry: Saez-Vasquez and Pikaard Proc. Natl. Acad. Sci. USA 94 (1997)

of protein for holoenzyme versus core polymerase due to the 17. Hempel, W. M., Cavanaugh, A. H., Hannan, R. D., Taylor, L. & larger mass of the former. Furthermore, less polymerase Rothblum, L. I. (1996) Mol. Cell. Biol. 16, 557–563. activity is recovered than is loaded onto each column, even 18. Bodeker, M., Cairns, C. & McStay, B. (1996) Mol. Cell. Biol. 16, when fractions are recombined, indicating that polymerase 5572–5578. activity decays throughout purification. Such lability negatively 19. Schnapp, G., Santori, F., Carles, C., Riva, M. & Grummt, I. (1994) EMBO J. 13, 190–199. affects specific activity and thus the estimated degree of 20. Schnapp, A., Pfleiderer, C., Rosenbauer, H. & Grummt, I. (1990) purification. We anticipate that antibodies specific for plant EMBO J. 9, 2857–2863. pol I subunits, currently unavailable, should allow us to better 21. Schnapp, G., Schnapp, A., Rosenbauer, H. & Grummt, I. (1994) follow pol I throughout purification and better estimate the EMBO J. 13, 4028–4035. abundance of the putative holoenzyme. 22. Yamashita, J., Nakajima, T., Tanifuji, S. & Kato, A. (1993) Plant J. 3, 187–190. We are grateful to Rob Larkin and Tom Guilfoyle (University of 23. Fan, H., Yakura, K., Miyanishi, M., Sugita, M. & Sugiura, M. Missouri) for generously providing antibodies against recombinant A. (1995) Plant J. 8, 295–298. thaliana RNA polymerase subunits and for sharing unpublished 24. Doelling, J. H., Gaudino, R. J. & Pikaard, C. S. (1993) Proc. Natl. results. We thank our fellow lab members Annie-Claude Albert, L. Acad. Sci. USA 90, 7528–7532. Annie Shen, and Milko Kermekchiev for their help and input. This 25. Doelling, J. H. & Pikaard, C. S. (1995) Plant J. 8, 683–692. work was supported by grants to C.S.P. from the NRI Competitive 26. Doelling, J. H. & Pikaard, C. S. 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