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Transcription Factor Requirement for Multiple Rounds of Initiation by Human RNA Polymerase II MARILYN N

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Transcription Factor Requirement for Multiple Rounds of Initiation by Human RNA Polymerase II MARILYN N Proc. Natl. Acad. Sci. USA Vol. 88, pp. 10691-10695, December 1991 Biochemistry Transcription factor requirement for multiple rounds of initiation by human RNA polymerase II MARILYN N. SZENTIRMAY AND MICHPLE SAWADOGO* Department of Molecular Genetics, University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030 Communicated by Donald D. Brown, September 3, 1991 ABSTRACT We have investigated conditions that allow (12, 13). Here we confirm that G-free cassette templates can multiple rounds oftranscription initiation from the adenovirus be used to directly distinguish first-round transcripts from major late promoter in an in vitro system derived from HeLa reinitiated transcripts in vitro. In addition, we present evi- cell nuclear extracts. Templates containing guanine-free cas- dence for the existence of a previously unknown transcrip- settes provided a direct assay for discriminating between tion factor, designated reinitiation transcription factor (RTF), reinitiated transcripts and transcripts generated by a first- which is absolutely required for efficient reinitiation by round of transcription initiations. When reactions were recon- human RNA polymerase II in vitro. stituted with the previously characterized class 11 transcription factors (TFIIA, TFIIB, TFIID, TFIIE/F), transcription by human RNA polymerase II from the adenovirus major late MATERIALS AND METHODS promoter was essentially restricted to a single round of initi- Purification of Transcription Factors and RNA Polymerase ations. Reinitiations at previously transcribed major late tem- HI. HeLa transcription factors TFIIB, TFIID, TFIIE, and plates required an additional activity, designated reinitiation RNA polymerase II were prepared essentially as described transcription factor (RTF). The RTF activity could be sepa- (8, 14). TFIIA and TFIIC [poly(ADP-ribose) polymerase] rated from the required transcription initiation factors. Semi- were also purified by published procedures (15, 16). purified human RTF also promoted transcription reinitiations Bacterially expressed human TFIID was purified from at minimal promoters derived from the human c-myc, histone Escherichia coli cells transformed with human TFIID cloned 114, and heat shock 70-kDa protein genes, indicating that the in a pET vector (a gift from M. C. Schmidt and A. J. Berk, same reinitiation factor may be utilized by many, if not all, University of California, Los Angeles). The recombinant genes. The possible role of RTF in regulating the transcription TFIID (3.3 units/ILI, 1.5 mg/ml) was purified from the soluble rate of various class 11 genes is discussed. E. coli extract by chromatography on DEAE-cellulose (DE- 52; Whatman) and Bio-Rex 70 (Bio-Rad). In contrast to prokaryotic RNA polymerases, human RNA RTF was separated from TFIIB by chromatography on a polymerase II alone cannot initiate transcription from pro- Mono S column (Pharmacia) in a buffer containing 20 mM moters but requires the participation of several general Hepes (pH 7.9 at 230C), 20% (vol/vol) glycerol, 0.2 mM factors (1). In vitro investigations of transcription from the EDTA, and 1 mM dithiothreitol (buffer A). Prior to this, the adenovirus major late (ML) promoter have revealed that the 0.5 M KCl fraction ofthe nuclear extract from Whatman P-11 first step in class II gene activation involves binding of class phosphocellulose purification was chromatographed on a II transcription factor D (TFIID) to the TATA element (2-5). DE-52 column. The breakthrough was loaded onto the Mono This initial interaction leads to template commitment and is S column in 100 mM KCl and was eluted with a gradient of sufficient to prevent promoter inactivation during nucleo- increasing KCl concentration in buffer A. RTF activity was some assembly (6). The committed template, the other gen- eluted in the 230 mM KCl gradient fraction, although signif- eral factors, and RNA polymerase II then assemble into a icant amounts of activity were also recovered in the column preinitiation complex (7). After transcription initiation and runthrough. TFIIB activity was reproducibly found in the promoter clearance by the RNA polymerase, a postinitiation Mono S 350 mM KCl gradient fraction. complex, containing TFIID and perhaps other components, Protein concentrations were determined by the method of remains at the promoter (8-10). The existence of this post- Bradford (17). initiation complex suggests that subsequent rounds of tran- In Vitro Transcription Assays. Transcription reaction mix- scription initiations (i.e., reinitiations) should proceed by a tures (25 ,pI) contained 50-70 mM KCl, 2-10 mM (NH4)2SO4, different mechanism than initial gene activation. At the least, 12 mM Tris (pH 7.3 at 250C), 12% glycerol, 3.6% (wt/vol) one might expect that the step in which TFIID binds to the polyethylene glycol 8000, 40 mM Hepes (pH 8.4), 7.5 mM template would not be necessary for subsequent initiations. MgCl2, 0.12 mM EDTA, 4 mM dithiothreitol, 0.6 mM ATP, Since this particular step appears to be generally rate-limiting 0.6 mM UTP, 25 AM [a-32P]CTP (5000-10,000 cpm/pmol), (3, 11), transcription reinitiations should be kinetically fa- and 8 units of RNasin (recombinant ribonuclease inhibitor; vored, unless specific mechanisms exist to control them. Promega). Unless otherwise noted, transcription reactions Experiments with various concentrations of sarkosyl to contained 0.3-1 unit of each of the general transcription inhibit individual reaction steps in RNA polymerase II tran- factors and RNA polymerase II and were incubated with 400 scription have shown that transcription reinitiations take ng of template for 45 min at 30'C (see ref. 14 for the definition place when the ML promoter is transcribed in crude HeLa of units). Reactions with linearized templates contained in nuclear extracts (11). Reinitiations were also invoked to addition 0.4 Iug of TFIIC to decrease possible nonspecific explain the pattern of decreasing-length transcripts some- initiations by RNA polymerase II (16). Reactions were times observed when templates containing guanosine-free stopped, extracted with 1:1 (vol/vol) phenol/CHC13, precip- (G-free) cassettes were transcribed in reconstituted reactions Abbreviations: TFIIA, TFIIB, etc., class II transcription factors A, The publication costs of this article were defrayed in part by page charge B, etc.; RTF, reinitiation transcription factor; ML, major late; payment. This article must therefore be hereby marked "advertisement" G-free, guanosine-free. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 10691 Downloaded by guest on September 27, 2021 10692 Biochemistry: Szentirmay and Sawadogo Proc. Natl. Acad. Sci. USA 88 (1991) itated with ethanol, and analyzed by electrophoresis through Smiai Pvui a 4.5% acrylamide gel as described (18). TATA +1 390 bp 0-less cassette The ribonucleoside triphosphates used in all transcription -, ... .. .:..I.... .. reactions were purified by chromatography on a Polyanion SI column (Pharmacia) to remove any contamination by GTP. However, it was necessary to add 0.1 mM 3'-O-methyl-GTP to reactions that included TFIIA because this fraction con- tained a small amount of endogenous GTP. First round of transcription RESULTS 901.,_t ranscrp G-free Cassette Templates Provide a Direct Assay for Tran- An_..:....... aiO3.9 nt transcrip r scription Reinitiation. Our analysis of transcription reinitia- tion relied on electrophoretic separation of transcripts initi- ated in vitro at a promoter cloned upstream of a G-free cassette (14). As illustrated schematically in Fig. 1 Upper, a ReEni tiations first-round of transcription initiation on such a template TFIID produces specific transcripts corresponding in length to the T-7-- cassette. In the absence of GTP, the RNA polymerase pauses, without disengaging, at the end of the G-free region --- shorter transcripts (8). Therefore, if transcription occurs a second time on the (390 nt, 360 nt, 330 nt, same template, the second RNA polymerase will be arrested TEMPL-ATE uncut PvuII SmWi by the presence of the first enzyme, producing RNA tran- scripts shorter by some 30 nucleotides (the length of DNA GTP CHASE - + covered by an elongating RNA polymerase molecule) (12). 1 h i 2 K- Similarly, a third round of initiation will give rise to tran- l:.... scripts that will be 60 nucleotides shorter than the full-length cassette, and so on. Our initial studies were carried out with a minimal pro- moter containing the TATA box and initiator region of the adenovirus ML promoter (from -50 to +10) upstream of a 4m 390-base-pair (bp) G-free cassette [plasmid pMLC2ATA-50 (12)]. As predicted in the above model, a regular pattern of decreasing-length transcripts was observed with this tem- plate when the basic factors required for in vitro transcription were provided by crude fractions from HeLa nuclear extracts or when the phosphocellulose 0.5 M KCI fraction was used as a source of TFIIB and TFIIE/F (Fig. 1 Lower). This pattern was present whether the DNA was circular (lane 1) or linearized (lane 3). FIG. 1. Assay for transcription reinitiations using G-free cassette were to that containing templates. (Upper) Schematic representation illustrating Several control experiments performed verify how first-round and reinitiated transcripts can be distinguished by the particular transcript pattern in these reactions was actu- their lengths when transcription elongation by RNA polymerase II is ally due to reinitiation events. If the shorter transcripts arrested at the end of the G-free cassette in the absence of GTP. resulted from RNA polymerases stacking at the end of the (Lower) Controls for the in vitro reinitiation assays. Transcription cassette, addition of GTP for a brief period of time at the end reactions were carried out as described with the 0.5 M KCl step of the reaction should allow transcript extension past the fraction from phosphocellulose chromatography as the source of G-free region. This was the case; after a chase with GTP, TFIIB and TFIIE/F. After a 25-min incubation at 30'C in the absence most of the full-length and reinitiated transcripts disappeared of GTP, transcription elongation was allowed to proceed for an (Fig.
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