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DNA-Dependent Atpase (Datpase) Activity Strongly Stimulated by the TATA Region of Promoters (Messenger RNA Synthesis/Core Promoter/Run-Off Transcription) RONALD C

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DNA-Dependent Atpase (Datpase) Activity Strongly Stimulated by the TATA Region of Promoters (Messenger RNA Synthesis/Core Promoter/Run-Off Transcription) RONALD C Proc. Natl. Acad. Sci. USA Vol. 86, pp. 7356-7360, October 1989 Biochemistry An RNA polymerase II transcription factor has an associated DNA-dependent ATPase (dATPase) activity strongly stimulated by the TATA region of promoters (messenger RNA synthesis/core promoter/run-off transcription) RONALD C. CONAWAY* AND JOAN WELIKY CONAWAY* Department of Chemistry and Clayton Foundation Biochemical Institute, University of Texas at Austin, Austin, TX 78712-1096 Communicated by Michael J. Chamberlin, June 26, 1989 (received for review May 11, 1989) ABSTRACT A transcription factor required for synthesis itate formation of the functional RNA polymerase II preini- of accurately initiated run-off transcripts by RNA polymerase tiation complex have not been defined. Finally, it has not II has been purified and shown to have an associated DNA- been possible to identify the factor or factors that mediate the dependent ATPase (dATPase) activity that is strongly stimu- ATP (dATP)-dependent activation of the preinitiation com- lated by the TATA region of promoters. This transcription plex or to determine how ATP is utilized in this activation factor, designated 8, was purified more than 3000-fold from step: whether it is hydrolyzed to provide energy or to serve extracts of crude rat liver nuclei and has a native molecular as a phosphate donor in a phosphorylation reaction or mass of approximately 230 kDa. DNA-dependent ATPase whether it is bound by a component of the transcription (dATPase) and transcription activities copurify when 8 is system. analyzed by hydrophobic interaction and ion-exchange HPLC, To address these questions, we sought to assemble a arguing that transcription factor 6 possesses an ATPase (dAT- transcription system composed of homogeneous RNA poly- Pase) activity. ATPase (dATPase) is specific for adenine nu- merase II and accessory factors. Thus far, we have purified cleotides; ATP and dATP, but not CTP, UTP, or GTP, are two accessory transcription factors, designated a and 8ry, to hydrolyzed. ATPase (dATPase) is stimulated by both double- apparent homogeneity and have demonstrated that they play stranded and single-stranded DNAs, including pUC18, ssM13, an integral role in formation of the functional preinitiation and poly(dT); however, DNA fragments containing the TATA complex (8, 11). We recently purified a third accessory region of either the adenovirus 2 major late or mouse inter- factor, designated 8, and found that it has an associated leukin 3 promoters stimulate ATPase as much as 10-fold more DNA-dependent ATPase (dATPase) activity. Further anal- effectively than DNA fragments containing nonpromoter se- ysis has led to the surprising discovery that this ATPase quences. These data suggest the intriguing possibility that 8 (dATPase) activity is strongly stimulated by the TATA region plays a critical role in the ATP (dATP)-dependent activation of of promoters. run-off transcription through a direct interaction with the TATA region of promoters. MATERIALS AND METHODS Initiation of mRNA synthesis is a key control point in the Materials. Male Sprague-Dawley rats (200-300 g) were from expression of many eukaryotic genes. Biochemical studies Simonson or Harlan-Sprague-Dawley. Unlabeled ultrapure have shown that initiation is an elaborate process requiring, ribonucleoside 5'-triphosphates and 2'-deoxynucleoside 5'- in addition to RNA polymerase II, multiple accessory tran- triphosphates were purchased from Pharmacia LKB Biotech- scription factors (1-12) and an ATP (dATP) cofactor (13-16). nology. [a-32P]CTP, [a-32P]ATP, [a-32P]GTP, [a-32P]UTP, Although the mechanism of initiation is at present poorly and [a-32P]dATP, all at 800 Ci/mmol (1 Ci = 37 GBq), were understood, a crude model has emerged from analyses of obtained from NEN. Bovine serum albumin (reagent grade) partially purified transcription systems. According to this was from ICN Immunobiologicals. Phenylmethylsulfonyl flu- model, one or more accessory factors first interact specifi- oride, antipain, and leupeptin were obtained from Sigma. cally with the TATA region of promoters. RNA polymerase Polyethylenimine-cellulose sheets were from Brinkmann. II, assisted by additional accessory factors, then recognizes Poly(dA), poly(dT), poly(C), poly(A), heparin, and single- and assembles with this nucleoprotein complex to form a stranded M13mpl8 were purchased from Sigma. functional preinitiation complex (4, 11, 17-21). Finally, in an Buffers. Buffer A was 20 mM Hepes (adjusted to pH 7.9 ATP (dATP)-dependent step, this complex is converted to an with "activated" complex, which is capable of initiating RNA NaOH)/1 mM EDTA/1 mM dithiothreitol/20% (vol/ synthesis rapidly upon addition of the remaining ribonucle- vol) glycerol/0.5 mM phenylmethylsulfonyl fluoride. Buffer oside triphosphates (16). C was 40 mM Tris-HCl, pH 7.9/0.5 mM EDTA/1 mM Although these studies have provided insight into the dithiothreitol/10% glycerol. Buffer D was 40 mM Hepes mechanism of initiation, an elucidation of the interactions (adjusted to pH 7.9 with NaOH)/0.5 mM EDTA/1 mM leading to formation and activation of the RNA polymerase dithiothreitol/10% glycerol. Buffer F was 40 mM Tris HCl, II preinitiation complex has been hindered by lack of a pH 7.5/0.5 mM EDTA/1 mM dithiothreitol/10% glycerol. purified transcription system. Several crucial questions re- Preparation of the Nuclear Extract. Fifty rats were lightly main unanswered. First, the exact number of accessory anesthetized with ether and killed by decapitation. All further factors required for initiation is not known; in fact, few ofthe steps were carried out at 4°C. The livers were removed, factors have been purified to homogeneity. Second, the rinsed in TMSD buffer (10 mM Tris HCl, pH 7.5/1.5 mM mechanisms by which the individual accessory factors facil- Abbreviation: Ad(-50 to +10), nucleotides -50 to +10 (relative to the cap site) of the adenovirus 2 major late promoter. The publication costs of this article were defrayed in part by page charge *Present address: Program in Molecular and Cell Biology, Oklahoma payment. This article must therefore be hereby marked "advertisement" Medical Research Foundation, 825 N.E. 13th Street, Oklahoma in accordance with 18 U.S.C. §1734 solely to indicate this fact. City, OK 73104. 7356 Downloaded by guest on October 3, 2021 Biochemistry: Conaway and Conaway Proc. Natl. Acad. Sci. USA 86 (1989) 7357 MgCl2/0.25 M sucrose/0.5 mM dithiothreitol/0.5 mM phen- DEAE-NPR HPLC column (35 x 4.6 mm) (Hewlett- ylmethylsulfonyl fluoride), minced, suspended in TMSD Packard) equilibrated with buffer C containing 0.05 M KCl. buffer to a final volume of 1500 ml, homogenized by one pass The column was eluted at 0.6 ml/min with a 9-ml linear through a continuous-flow homogenizer (22), and centrifuged gradient from 0.05 M to 0.27 M KCl, and 0.2-ml fractions at 800 x g for 10 min. The crude nuclear pellet was washed were collected. twice by resuspension in TMSD buffer and centrifugation at Preparation of RNA Polymerase II and Transcription Fac- 800 x g for 10 min, suspended in TMSD buffer to 2000 ml, and tors. RNA polymerase II and transcription factors a and fry extracted with 0.33 M (NH4)2SO4 by dropwise addition of 180 were purified from rat liver as previously described (8, 11). ml of saturated (NH4)2SO4 with gentle stirring (see Fig. 1). Fraction D was purified from the nuclear extract by phos- After 30 min, the extract was centrifuged at 12,000 X g for 90 phocellulose and carboxymethyl-Sephadex chromatography min. Solid (NH4)2SO4 was then added slowly to the super- performed as described (8). Active fractions from carboxy- natant (fraction I; see Table 1) to 40% saturation [0.186 g of methyl-Sephadex were concentrated by precipitation with (NH4)2SO4 per ml]. After the addition of 1 ,l of1M NaOH per ammonium sulfate (0.35 g/ml) and further purified by gel g of (NH4)2SO4, the suspension was centrifuged at 12,000 x filtration on a 1.5- x 60-cm AcA 22 column (IBF Biotechnics) g for 45 min. The precipitate was resuspended in buffer A in buffer A containing 0.5 M KCl and by chromatography on containing leupeptin and antipain at 10 ,ug/ml each and a 35- x 4.6-mm TSK DEAE-NPR HPLC column, which was dialyzed against buffer A to a conductivity equivalent to 0.1 eluted at 0.6 ml/min with a 9-ml gradient from 100 to 400 mM M KCI (fraction II). KCl in buffer C. Purification of Transcription Factor 6. Fraction II was Assay of Run-off Transcription. Except when indicated in centrifuged at 4000 x g for 10 min and then loaded onto a the figure legends, assays were performed as described (8) 100-ml phosphocellulose column (P11, Whatman) equili- with 0.1 /ig of Nde I-digested pDN-AdML (16), 50 ng of brated with buffer A containing 0.1 M KCl. Transcription fraction D, 2 ng of transcription factor a (fraction V), 10 ng activity was eluted stepwise at one packed column volume oftranscription factor ,8y (fraction V), and 0.01 units ofRNA per hour with buffer A containing 0.5 M KCl. One-fifth polymerase II. Reaction mixtures contained ATP, UTP, and column volume fractions were collected, and the active GTP at 50 tLM, CTP at 10 ,uM, and 10 kLCi of [a-32P]CTP; fractions were pooled and dialyzed against buffer C to a heparin (10 pgg/ml) was routinely added to reaction mixtures conductivity equivalent to buffer C containing 0.05 M KCl 2 min after addition of the four ribonucleoside triphosphates (fraction III). Fraction III was centrifuged at 16,000 x g for and magnesium in order to limit transcription to one round of 20 min and applied to a Spherogel TSK DEAE-SPW column initiation per promoter (10).
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