THE OF CHEMISTRY Vol. 266, No. 36, Issue of 25, pp. 24588 ...THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc Vol. 266,

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 266, No. 36, Issue of December 25, pp. 24588-24595,1991 Printed in U. S. A .

Transcription Complex Formation at the Mouse rDNA Promoter Involves the Stepwise Association of Four Transcription Factors and RNA Polymerase I*

(Received for publication, July 8, 1991)

Andreas Schnapp and Ingrid Grummt From the Institute of Cell and Tumor Biology, German Cancer Research Center, 0-6900 Heidelberg, Federal Republic of Germany

We have used purified transcription factors and RNA polymerase I (pol I) to analyze the individual steps involved in the formation of transcription initiation complexes at the mouse ribosomal gene promoter in vitro. Complete assembly of transcription complexes requires pol I and at least four auxilliary factors, termed TIF-IA, TIF-IB, TIF-IC, and UBF. Preincuba- tion and template commitment, as well as order of addition protocols, were used to discriminate between various intermediate complexes generated during assembly of the initiation complex. As a first step, TIF- IB binds to the core promoter, a process that is facilitated by the upstream control element and the upstream binding factor (UBF). Binding of TIF-IB to the rDNA promoter results in the formation of a functional preinitiation complex (complex l), which is stable for many rounds of transcription. UBF,which on its own does not stably associate with the rDNA promoter, triggers a 5-10-fold increase in the overall amount of this primary complex. Following binding of TIF-IB and UBF to the template DNA, pol I and TIF-IC successively bind,yielding complexes 2 and 3, respectively. Transcription-competent initiation complexes are built up by the final association of the growth-regulated factor TIF-IA. The various complexes can be distin- guished by their different sensitivity to Sarkosyl. Only the complete complex consisting of all four factors and pol I shows resistance to intermediate concentrations of Sarkosyl (0.045%) and is competent to catalyze the formation of the first phosphodiester bond. The initiated complex is, on the other hand, resistant to high concentrations of Sarkosyl (0.3%). The hierarchical nature of the different complexes formed suggests a model for transcription initiation and predicts functions for the individual factors.

The agglomeration of multiple proteins, some of which bind t o specific sites in DNA, is a common theme in eukaryotic gene regulation. For all three classes of RNA polymerases, specific transcription factors have been shown to assemble at the promoter in an ordered fashion into multiprotein-DNA complexes, a process that is a prerequisite for specific transcription initiation (for review, see Grummt (1989), Sawadogo and Sentenac (1990), Palmer and Folk (1990)). A thorough

*This work was supported by the Deutsche Forschungsgemein- schaft, the Fonds der Chemischen Industrie, and Grant SCI*-O259-C of the Science Program of the Commission of the European Com- munity. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

understanding of promoter recognition in eukaryotic cells requires both the identification of the trans-activators involved and a detailed mechanistic insight of how these factors interact with the DNA, with each other, and with the RNA polymerase.

For eukaryotic RNA polymerase I1 (pol 11)’ transcription, a number of systematic studies has been carried out to define an ordered pathway of preinitiation complex formation (Bur- atowski et al., 1989; Conaway et al., 1990; Conaway and Conaway, 1990; Maldonado et al., 1990). Based on kinetic analyses, on the resolution of different template-protein complexes by electrophoresis in native gels, or by using template commitment protocols, a model has been proposed that describes different steps and the involvement of different general transcription factors in the overall pathway. Apparently, TFIIA acts at an early step of complex formation, presumably by facilitating binding of TFIID to the TATA element (Sa- wadogo and Roeder, 1984; Nakajima et ul., 1988; Van Dyke et al., 1989; Conaway and Conaway, 1989a). The complex formed between TFIID and TFIIA mediates the subsequent association of TFIIB (Buratowski et al., 1989; Maldonado et al., 1990). After binding of TFIIB, the polymerase associates with the preinitiation complex (Buratowski et al., 1989). Binding of pol I1 in turn is necessary for the association of factors TFIIE and TFIIF. Both TFIIE and TFIIF are general pol I1 transcription initiation factors that have been shown to spe- cifically interact with RNA polymerase I1 (Flores et al., 1988; Flores et al., 1989; Inostroza et al., 1991).

Although multiple nucleolar transcription factors have been identified that work together with pol I in promoter recognition, much less is known about the exact order of assembly and the mechanisms of action of the various factors that govern initiation of ribosomal RNA gene transcription. Sim- ilar to pol 11, class I RNA polymerase will not accurately initiate transcription in vitro unless reconstituted with additional factors. We have shown that in addition to pol I, four activities are required to reconstitute specific transcription initiation at the mouse rDNA promoter (Schnapp et al., 1991). These factors have been partially purified and designated in the mouse system TIF-IA (Buttgereit et al., 1985; Schnapp et al., 1990b), TIF-IB (Clos et al., 1986a, 1986b; Schnapp et d., 1990a). TIF-IC,’ and UBF (Bell et al., 1990). Factor TIF-IA,

The abbreviations used are: pol 11, DNA-dependent RNA polymerase 11; TFII, transcription factor of RNA polymerase 11; TIF, transcription initiation factor; pol I, DNA-dependent RNA polymerase I; UBF, upstream binding factor; FPLC, fast protein liquid chromatography; AMP-PNP, adenyl-5”yl imidodiphosphate; araATP, adenine-9-fl-D-arabinofuranoside 5”triphosphate; ATP-& adenosine 5’-O-(thiotriphosphate).

2 H . Rosenbauer, G. Heilgenthal, A. Schnapp, and I. Grummt, manuscript in preparation.

24588

Transcription Complex Formation 24589

also known as TFIC (Mishima et al., 1982; Mahajan and Thompson, 1990) or factor C (Tower and Sollner-Webb, 1987) has been shown to interact with pol I (Schnapp et al., 199Ob). Interestingly, the amount or activity of TIF-IA correlates with cell proliferation and, therefore, this factor appears to play a key role in regulation of ribosomal gene transcription.

The mouse rDNA promoter is recognized by two sequence- specific DNA-binding proteins, TIF-IB (Clos et al., 1986a, 1986b; Schnapp et al., 1990a) and the upstream binding factor UBF (Learned et al., 1986). TIF-IB, the factor that other groups designate factor D or TFID (Mishima et al., 1982; Tower et al., 1986; Tanaka et al., 1990) or mSLl (Bell et al., 1990), binds to the core promoter and confers promoter selec- tivity to pol 1. TIF-IB or its homolog in other organisms is the protein responsible for the observed species specificity of rDNA transcription (Grummt et al., 1982; Onishi et al., 1984; Learned et al., 1985; Bell et al., 1990; Schnapp et al., 1991). Binding of TIF-IB to its target sequence appears to be a prerequisite for the assembly of the other factors and pol I into a functional transcription initiation complex.

Although our previous studies indicated that both TIF-IA and TIF-IC interact with pol I and perform essential functions in rDNA-specific transcription, numerous questions remained concerning the mechanism by which pol I and these non- DNA-binding factors assemble at the promoter. This paper describes a series of experiments designed to investigate how each of these factors function in initiation, in particular at which step in the initiation reaction the individual factors promote formation of distinct intermediates in the pathway leading to assembly of an active initiation complex. Using both highly or partially purified transcription factors, we demonstrate a hierarchy of specific protein-promoter complexes. Each complex requires a specific set of transcription factors for formation and reacts to inhibitor challenges in a manner consistent with previous studies on the formation of pol I transcription initiation complexes. Kinetic and template commitment experiments indicate that all four factors and pol 1 become stably associated with the rDNA promoter in an ordered fashion and that all factors act in a stage prior to transcription initiation.

EXPERIMENTAL PROCEDURES

Materials-Ultrapure ribonucleoside 5’-triphosphates were pur- chased from Pharmacia LKB Biotechnology Inc. AMP-PNP was obtained from Sigma.

rDNA Templates-The template pMrSP contains the 5”terminal SalI/PuuII fragment cloned into pUC9 from mouse rDNA extending from nucleotides -168 to +292 with respect to the transcription initiation site. Plasmid pMrWT contains a 323 base pair SalI/SmaI fragment from position -168 to +155 (Skinner et al., 1984). pMrSP was cleaved with EcoRI and pMrWT with NdeI to generate 297- and 379-nucleotide run-off transcripts, respectively.

Cell Culture and Extract Preparation-Mouse Ehrlich ascites cells were cultured in RPMI medium supplemented with 5% newborn calf serum for 20-40 h. Transcriptionally active extracts were prepared from a logarithmically growing cell culture (9 X IO5 cells/ml). S-100 extracts were prepared according to Weil et al. (1979) and nuclear extracts according to Dignam et al. (1983).

Purification of RNA Polymerase I and Transcription Initiation Factors-A typical factor preparation started from about 300 ml (1.8 g of protein) of a mixture of nuclear and cytoplasmatic extracts prepared from cultured Ehrlich ascites cells. Part of the fractionation procedure has been published (Schnapp et al., 1990a, 1990b). Buffers used were either buffer A (20 mM Tris-HC1, pH 7.9, 0.1 mM EDTA, 20% glycerol, 0.5 mM dithioerythritol and 0.5 mM phenylmethylsulfonyl fluoride) or buffer AM (same as buffer A, but with 5 mM MgC12) containing different concentrations of KC1. As a first step, extracts were fractionated on a DEAE-Sepharose CL-GB column, and bound proteins were step-eluted at 280 mM KC1. After dilution to 200 mM KCI, the DEAE-280 fraction was loaded onto a heparin-Ultrogel A4R

column according to method 2 in Schnapp et al. (1990b). Bound proteins were eluted successively with 200, 250, 400, 600, and 1000 mM KC1 in buffer AM, yielding fractions H-200, H-250, H-400, H- 600, and H-1000, respectively. Factors TIF-IA and TIF-IC present in the H-200 fraction were applied to a Q-Sepharose column, and both activities were eluted at 300 mM KCl. Subsequently, TIF-IA was separated from TIF-IC by high performance liquid chromatography on a polyethyleneimine high performance liquid chromatography column. Bound proteins were eluted with a linear gradient from 100 to 1000 mM KCI, with the peak of TIF-IC activity eluting at about 550 mM KCl, and TIF-IA activity eluting at about 700 mM KCl. Both TIF-IC and TIF-IA were further purified by chromatography on a Mono-Q FPLC column. Bound proteins were recovered with a linear salt gradient from 200 to 300 mM KCI, with both activities eluting at about 230 mM KCI. The protein concentration of TIF-IA- or TIF-IC- containing fractions was about 75 and 150 pg/ml, respectively.

The H-400 fraction contained RNA polymerase I and varying amounts of UBF, TIF-IC, and TIF-IA. RNA polyermase I was further purified and separated from these contaminating activities by gradient elution (150-400 mM KCl) from a Mono-Q FPLC column. The peak of activity that eluted at 250 mM KC1 was applied to a Mono-S FPLC column, and bound proteins were recovered with a linear salt gradient ranging from 200 to 500 mM KCI. pol I eluted at 350 mM KCI. RNA polymerase I preparations used in this work were not homogeneous but did not contain measurable levels of TIF-IA, TIF- IC, or UBF activity. TIF-IB and contaminating UBF were recovered in the H-600 fraction and purified further by chromatography on CM-Sepharose, as described by Schnapp et al. (1990a). On this column, UBF and TIF-IB were step-eluted at 200 and 400 mM KCI, respectively. Further purification of TIF-IB (CM-400 step) was achieved by fast performance liquid chromatography on a Mono-Q FPLC column. Bound proteins were eluted with a linear gradient from 150 to 400 mM KCI, with TIF-IB eluting at about 230 mM KCI. TIF-IB purified according to this procedure was free of contaminating UBF as determined (i) by the absence of UBF activity, (ii) by the absence of UBF bands in silver-stained gels, and (iii) by phosphorylation of TIF-IB fraction with casein kinase 11, for which UBF is an excellent substrate in ~ i t r o . ~ The protein content of the TIF-IB fraction was about 10 pg/ml.

UBF was purified from the H-1000 fraction to apparent homoge- neity by chromatography on a Mono-Q FPLC column, followed by a sequence-specific DNA affinity c01umn.~

In Vitro Transcription Assays-Transcription reactions and pro- duct analysis were performed as described before (Grummt, 1981; Clos et al., 1986a, 1986b). Preincubation of either pol I or the individual factor preparations with the template DNA was performed under standard transcription conditions (12 mM Tris, pH 7.9, 0.1 mM EDTA, 5 mM MgCI2, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithioerythritol, 80 mM KCl, and 12% glycerol (v/v)) in the absence of nucleoside triphosphates at 30 “C. At the times indicated in the figure legends, transcription reactions were initiated by the addition of the missing factor(s) and nucleoside triphosphates (0.66 mM each of ATP, CTP, and UTP, 12.5 ~ L M GTP, and 3 pCi [a3’P]GTP (400 &i/mmol)). When indicated, Sarkosyl was present during the preincubation or elongation period at the concentrations depicted in the figure legends.

RESULTS

Four Transcription Initiation Factors, TIF-IA, TIF-IB, TIF-IC, and UBF, Promote Formation of a Transcription- competent Complex-Reagents such as heparin and Sarkosyl have been used to analyze different steps in the formation of transcription initiation complexes. Both for class I and I1 genes, the sensitivity of transcription against these inhibitors has been shown to reflect distinct multiprotein complexes assembled at the promoter (Hawley and Roeder, 1985; Kat0 et al., 1986; Reinberg et al., 1987; Reinberg and Roeder, 1987; Flores et al., 1989; Conaway et al., 1990; Gokal et al., 1990). In this study, we have used purified transcription factors and partially purified pol I to analyze the individual steps involved in the formation of transcription initiation complexes at the murine ribosomal gene promoter. In an initial set of experi-

A. Schnapp, data not shown. ‘ H. Rosenbauer, data not shown.

24590 Transcription Complex Formation

ments, we have determined the stability of various complexes in the presence of Sarkosyl (data not shown). In agreement with similar studies performed by Kat0 et al. (1986), we found that 0.045% of Sarkosyl completely inhibits rDNA transcrip" tion when added to the assay before or simultaneously with RNA polymerase I and the four auxiliary factors, TIF-IA, TIF-IB, TIF-IC, and UBF (Fig. LA, lane 2). However, preincubation of the template with RNA polymerase I and all factors before adding the inhibitor renders transcription resistant to 0.045% of Sarkosyl (lune 3).

These findings are consistent with the hypothesis that Sarkosyl inhibits reactions prior to transcription initiation but does not affect elongation. The low transcription signal in lane 3 is due to the fact that Sarkosyl inhibits the formation of the preinitiation complex, and reinitiation does not occur in the presence of this reagent. Consequently, each functional promoter complex gives rise to only one transcript, whereas in the absence of this inhibitor, pol I reinitiates and generates several transcripts per template (lane 1). To determine which protein fractions have to be present during the preincubation period to yield Sarkosyl-resistant preinitiation complexes, either of the five fractions was omitted from the preincubation mixtures. After preincubation for 30 min, Sarkosyl was added, and transcription was started by addition of nucleoside triphosphates and the missing factor (Fig. 1B). Sarkosyl-resist- an t complexes were only formed when all factors, pol I, and the template DNA were present during the preincubation period (lane 7). No signal (except in lane 2, which shows a weak signal after longer exposure) was observed in the absence of any factor or pol I, a finding that implies a function of all five protein fractions in reactions preceding initiation of rRNA synthesis. Fig. 1C illustrates the kinetics of complex formation. Consistent with previous data showing a lag phase in rDNA transcription initiation (Wandelt and Grummt, 1983; Kat0 et al., 1986), the formation of preinitiation complexes is a slow process. The first visible signal was observed after preincubating all factors with the rDNA template for 10 min (lane 4) . Under the experimental conditions employed,

A B

complex formation was completed after 20 min (lane 6). Longer preincubation periods did not result in stronger transcription signals.

A Committed Complex Is Formed by the Association of TIF- IB with the rDNA Promoter-To investigate the exact order according to which the individual proteins stepwise associate with the rDNA promoter and thus form a functional preinitiation complex, template exclusion experiments were performed (Bogenhagen et al., 1982; Wandelt and Grummt, 1983). In this assay, two different rDNA templates were used. Template 1 was pMrSP, a plasmid containing mouse rDNA sequences from -168 to +292 (see "Experimental Proce- dures"). Template 2 was pMrWT, encompassing rDNA gene sequences from -168 to +155 (Skinner et al., 1984). The templates were linearized with EcoRI or NdeI to yield 297- and 379-nucleotide run-off transcripts, respectively. When equimolar amounts of both DNAs were present during the preincubation period, both templates were transcribed with equal efficiency (Fig. 2A, lane 1). Preincubation of either template with a given amount of the individual protein fractions precludes transcription of the subsequently added second template (lanes 2 and 3), indicating that there is a stable association of factors with the first template. Formation of stable preinitiation complexes a t rDNA promoters has been reported before (Wandelt and Grummt, 1983; Cizewski and Sollner-Webb, 1983; Clos et al., 1986a; Kat0 et al., 1986; Tower et ul., 1986; Mahajan and Thompson, 1990). However, those experiments were performed either with unfractionated extracts or with crude fractions. The availability of purified factor preparations, each of which plays an essential role in the initiation process, enabled us now to use template commitment assays to study the defined order according to which the individual factors assemble into the initiation complex.

As a first step, we investigated whether association of the two DNA-binding proteins TIF-IB and UBF with the mouse rDNA promoter are sufficient to form a stable preinitiation complex that resists challenge by the second template. For this, limiting amounts of factor TIF-IB, UBF, or a combina-

C & 3 - 3 ; m 4 0

preincubation - + - transcription - + + Sarkosyl - + + + + + + preincubation 20 0 5 10 15 20 25 min

missing protein - 3 X I- F - Sarkwyl - + + + + + + Sarkosyl in

e -

1 2 3 1 2 3 4 5 6 7 1 2 3 4 5 6 7

FIG. 1. Formation of a Sarkosyl-resistant complex in the reconstituted transcription system. A, formation of an initiation complex resistant to 0.045% of Sarkosyl. RNA polymerase I (3 pl) , TIF-IA (3 pl), TIF- IB (3 pl), TIF-IC (3 pl), and UBF (1 p l ) were preincubated for 30 min at 30 "C with 50 ng of template pMrSP/ EcoRI in a volume of 21 pl in the absence (lanes 1 and 3) or in the presence (lane 2) of 0.045% of Sarkosyl. Transcription was initiated by addition of nucleoside triphosphates. In lane 3, Sarkosyl was added to a final concentration of 0.045% together with the NTPs. Transcription reactions were stopped after 30 min. B, four auxilliary factors and RNA polymerase I are involved in the formation of the transcription initiation complex. 50 ng of template DNA pMrSP/EcoRI were preincubated for 30 min at 30 "C with pol I and the four transcription initiation factors (lanes 1 and 7). In the reactions shown in lanes 2-6, either one of the factors or pol I were omitted from the preincubation assays. Transcription was initiated by addition of the missing proteins and nucleoside triphosphates and was carried out for 20 min at 30 "C in the presence of 0.045% Sarkosyl (except lane 1 ). C, time course of transcription initiation complex formation. RNA polymerase I (24 pl) , TIF-IA (24 pl), TIF-IB (24 pl), TIF-IC (24 pl), and UBF (8 pl) were incubated with 400 ng of template pMrSP/EcoRI at 30 "C in a total volume of 168 pl. At the times indicated aboue the lanes, 23-p1 aliquots of the preincubation mixture were removed, Sarkosyl was added to a final concentration of 0.045%, and transcription was started by addition of nucleoside triphosphates. Transcription reactions were performed for 20 min at 30 "C.


B C

presenlduringplna~ba(ion

A

, . TIF-IB + + + + - - UBF - - + + + +

template2 + - + template2 - + - + - + template 1 + + - template1 + - + - + - preinwbation(min) 0 1 3 5 10

379m

297 m 297 m

1 2 3 1 2 3 4 5 6 1 2 3 4 5

pnincubasm lrar*w*bon / # \ / ' * \ uu: l*lanwm T P"dMnlp4ale x' I stop I

X 4 C

1alBwm Z?!*np(.* nF.m& rnopnpmeim U8F N T R UBF TIF4MlF.K:

nF.m poll

NTP?I

FIG. 2. Formation of a template-committed complex by factor TIF-IB. A, template exclusion experiment in the reconstituted transcription system. Lane 1, purified pol I (3 pl) , TIF-IA (3 pl) , TIF-IB (1 pl), TIF-IC (3 PI) , and UBF (0.5 pl ) were incubated with 25 ng each of the templates pMrSP/EcoRI and pMrWT/NdeI for 20 min at 30 "C prior to the addition of nucleotides (NTPs). In lanes 2 and 3, either pMrSP/EcoRI (lane 2) or pMrWT/ NdeI (lane 3) were incubated with pol I and factors for 20 min prior to the addition of the second template and NTPs. Transcription reactions were performed for 40 min. B, formation of stable preinitiation complexes in the presence of TIf-IB and UBF. 25 ng of the first template (either pMrSP/EcoRI or pMrWT/NdeI) were preincubated with either TIF-IB (1 pl) or UBF (0.5 pl) or a mixture of both factors in a volume of 8 pl. 25 ng of the second template were added together with pol I (3 pl) , TIF-IA (3 pl), TIF-IC (3 pl), nucleoside triphosphates (NTPs) , and if necessary, TIF-IB or UBF. Transcription reactions were stopped after 40 min. C, time course of assembly of complex 1. 125 ng of template pMrWT/NdeI were incubated with TIF-IB (5 pl) and UBF (2.5 pl) at 30 "C in a total volume of 40 pl. At the times indicated above the lanes, 25 ng of template DNA pMrSP/EcoRI, pol I (3 PI), TIF-IA (3 pl), TIF-IC (3 pl), and NTPs were added in a volume of 17 pl to 8-pl aliquots of the preincubation mixture. Transcription reactions were stopped after 40 min. X , length of the preincubation period (0, 1, 3, 5, or 10 min). nt, nucleotides.

tion of both factors were incubated for 20 min with either template 1 or template 2. Reaction mixtures were completed by addition of the second DNA, RNA polymerase I, TIF-IA, TIF-IC, and nucleoside triphosphates. Preferential transcription of the first template, which is diagnostic for the formation of stable complexes, was observed only in the reactions where TIF-IB was present during the preincubation period (Fig. 2B, lanes 1-4). Apparently, TIF-IB on its own stably binds to the mouse rDNA promoter (lanes 1 and 2), whereas UBF does not (lanes 5 and 6). However, preincubation of both TIF-IB and UBF together consistently resulted in more efficient transcription of the first template (lanes 3 and 4) , indicating a cooperative interaction between TIF-IB and UBF during preinitiation complex formation. Nevertheless, the exclusive transcription of the first template after incubation with highly purified TIF-IB led us to conclude that TIF-IB forms a committed complex independent of the presence of the other factors. Thus the TIF-IB-promoter complex appears to be the first step in the assembly of a functional preinitiation complex and is designated complex 1. Once formed, this complex is very stable. It remains bound to its target DNA even after prolonged incubation (for 90 min) with a second template and resists 0.005% Sarkosyl (data not shown).

Next, we addressed the question whether formation of complex 1 is the rate-limiting step in the assembly reaction. The first template (pMrWT/NdeI) was incubated with limiting amounts of TIF-IB and UBF for 1, 3, 5, and 10 min before the second template (pMrSp/EcoRI), RNA polymerase I, TIF-IA, TIF-IC, and nucleotides were added. The data shown in Fig. 2C demonstrate that formation of complex 1 is

a very rapid process. Preferential transcription of the first template is already observed after preincubation for only 1 min (lane 2). After 2-3 min, the association of TIF-IB (and probably UBF) with the promoter is complete (lanes 3-5). Therefore, the lag phase of about 20 min required to assemble intact preinitiation complexes (see above) must be due to later steps of complex formation, i.e. association of pol I, TIF-IA, or TIF-IC, respectively, with complex 1.

Stepwise Association of RNA Polymerase I, TIF-IA, and TIF-IC with Complex I-To investigate in which order TIF- IA, TIF-IC, and RNA polymerase I join the primary DNA- protein complex, the template commitment assay was combined with an order of addition protocol as diagramed in Fig. 3. Briefly, two separate reactions containing either template 1 (pMrSPIEcoR1) or template 2 (pMrWT/NdeI) were preincubated for 10 min with the factor combinations indicated to preassemble initial complexes (preincubation 1). As a next step, subsaturating amounts of pol I, TIF-IA, or TIF-IC were added to one of the two reactions and allowed to interact with the primary or intermediate complexes for another 10 min (preincubation 2). Then, both reactions were combined and the missing factors (if any) were added. After 10 min (preincubation 3), transcription reactions were initiated by the addition of nucleotides in the presence of 0.045% Sarkosyl. If stable intermediate complexes were assembled either on template 1 or on template 2 during the first two preincubation periods, transcripts should preferentially initiate from the promoter of this template. If, however, proteins act catalyti- cally, or interact not at all with the complexes formed during the first preincubation period, equivalent amounts of run-off


Prelncuballon 1 RNA Prelncubation 3 PrelnCubaHon 2

template +proteins lane 2 +proteins +proteins

template t - template 2 p o l

template 1 pol template 2 - template 1 BN - template 2 pol

template 2 B/U template 1 B/U POI

template 1 B/U template 2 B/U

template 1 B/U template 2 B/U

template 1 B N / ~ I - template 2 C A template 1 pol template2 ~ ~ l p o l - template I B N / ~ I - template 2 B N l m l A

- BNIAx: -

- - BNIAx:

Ax:

Ax: - -

C poVA

C POVA -

C A

C

I template 1 B ~ l p o l template 2 BNIDol ternplate 1 ~ ~ l p o l ~ : - template2 B ~ l p o l l C A

template 1 BNlpoVC A template2 BN/~~ I /C -

- -

* I 9 1

9

Preincubation 1 Preincubation 2 Preincubalon 3 Transcription

/ V V V \

template 7') 1: +proteins pMrSPEmRl +proteins Io' I 5' s l~p

template 2: pMrWMdel +proteins

lo' + proteins +MPs +Sarkosyl

+proteins

FIG. 3. Stepwise association of pol I, TIF-IC, and TIF-IA with complex 1. Two separate reactions containing either 40 ng of the template pMrSP/EcoRI (template 1) or pMrWT/NdeI (template 2) were preincubated for 10 min with the protein fractions indicated in the diagram. In the next step, subsaturating amounts of either pol I, TIF-IA, or TIF-IC were added to one of the reactions, and incubation was continued for another 10 min (preincubation 2). Then the two reactions were combined, the missing factors were added, and after 10 min (preincubation 3), transcription was started by addition of NTPs. The order of addition of the individual factors is diagramed in the figure. During the elongation period, Sarkosyl was present a t a final concentration of 0.045%. A single assay contained 1 pl of RNA polymerase I (pol), 0.5 pl of factor TIF-IA ( A ) , 2 p1 of factor TIF-IB (R), 0.5 p1 of factor TIF-IC (C), and 1 pl of UBF ( U ) .

transcripts initiated from both promoters should be synthesized.

The data shown in Fig. 3 provide the following information. (i) pol I, which on its own does not stably bind to the template DNA (lanes 1 and 2) is recruited to the promoter by DNA- bound TIF-IB/UBF (lanes 3 and 4 ) . The complex formed by association of pol I with the TIF-IB/UBF-promoter complex is termed complex 2. (ii) Both TIF-IA and TIF-IC do not stably associate with the DNA template in the absence of pol I (lanes 5 and 6)5 but interact stably and stoichiometrically with complex 2. (iii) TIF-IA and TIF-IC assemble with the preinitiation complex in a preferred order. In the absence of TIF-IA, TIF-IC is capable of promoting the formation of a stable intermediate complex (lunes 7 and 8). In the absence of TIF-IC, however, TIF-IA does not interact with complex 2 (lanes 9 and 10). Assembly of TIF-IC in turn enables TIF-IA to enter the complex (lanes 11 and 12). As neither TIF-IA nor TIF-IC is capable of interacting stably with DNA or with each other,' it is likely that these factors interact with complex 2 via specific contacts with the polymerase to generate complexes 3 and 4, respectively.

'A. Schnapp and I. Gmmmt, unpublished data.

Formution of the Initiated Complex-The following experiments were performed to study the conversion of the preinitiation complex (complex 4) into an initiated complex that is capable of elongation in the presence of high concentrations (0.3%) of Sarkosyl. Formation of such initiated complexes requires the first and second nucleotides of mouse pre-rRNA (ATP and CTP) to allow the formation of the first phosphodiester bond (Kato et al., 1986; Gokal et al., 1990). Preinitia- tion complexes were assembled on the template pMrSP/ EcoRI either in the presence or absence of ATP plus CTP, and transcription reactions were then performed in the presence of two concentrations of Sarkosyl (0.045% and 0.3%). The data shown in Fig. 4 are consistent with previous findings demonstrating that only complexes formed in the presence of ATP and CTP were transcriptionally active in the presence of 0.3% Sarkosyl (compare lane 3 with lane 6). ATP and CTP alone or other nucleotide combinations did not substitute for ATP and CTP (data not shown). Although this result shows that formation of the first internucleotide bond is responsible for this Sarkosyl-resistant transcription, it does not rule out the possibility that an energy-requiring process involving ATP hydrolysis contributes to conversion of the preinitiation to the initiation complex. We therefore substituted ATP by AMP-PNP in the preincubation mixture and asked whether or not this nonhydrolyzable ATP analogue would also confer detergent resistance to the initiated complexes. There are no significant differences in the level of transcripts synthesized in the presence of ATP or AMP-PNP, respectively (compare lanes 4-6 with lanes 7-9), indicating that formation of the initiated complex does not require ATP hydrolysis. Since AMP-PNP does not exert an inhibitory effect on rDNA transcription, it is reasonable to conclude that neither initiation nor elongation by pol I requires an ATPase activity.

DISCUSSION

In this study, we have analyzed the assembly of functional preinitiation complexes at the mouse ribosomal gene promoter in an attempt to define the ordered association of transcription initiation factors and pol I at the rDNA template. These studies were facilitated by the availability of a reconstituted

Y) * Y) -$

- 0 0 Sarkosyl (X) - 2 2 - 0 0 3 9

nudeotides present during preincubation - - -

9 -

1 2 3 4 5 6 7 0 9

FIG. 4. Formation of the initiated complex. Transcription reactions containing RNA polymerase I, TIF-IA, TIF-IB, TIF-IC, and UBF were preincubated with 50 ng of template pMrSP/EcoRI at 30 "C. Preincubation reactions were performed either in the absence of nucleotides (lunes 1-3) or in the presence of 0.66 mM each of ATP and CTP (lunes 4-6) or AMP-PNP and CTP (lanes 7-9). After 20 min, Sarkosyl was added to the final concentrations indicated above the lunes. After an additional 10 min of incubation, transcription reactions were initiated by the addition of the missing nucleotides. Reactions were stopped after 5 min. The same amounts of proteins as depicted in Fig. 1 were used.


transcription system containing four transcription initiation factors and pol I, each of which has been purified by at least four chromatographic columns and did not show a cross- contamination with any of the other protein components. We have demonstrated a hierarchy of specific protein-promoter complexes that assemble at the rDNA promoter in an ordered fashion and provided experimental evidence that all factors play an indispensable role in a stage prior to transcription initiation. Nevertheless, our data do not exclude the possibility that in addition to their pivotal role in initiation, any of these factors may also be required in the elongation phase of transcription.

A schematic representation of the composition of the intermediate multiprotein complexes assembled at the rDNA promoter, as well as the order of association of the individual factors with the rDNA promoter, is shown in Fig. 5. The first step in initiation is the recognition of the rDNA promoter by TIF-IB, a process that is inhibited by low concentrations of Sarkosyl (0.005%) and is 5-10-fold enhanced by UBF. Once TIF-IB is bound, it remains stably associated with the tem-

-40 +1

* 1DNA

1 * complex 1 A*

IF48 UBF complex 2

1

& *

TIF-IC

complex 3

Sarkosyl resistance

0.005%

0.005%

n.d

complex 5 (initiated complex) 0.3% I

'

+ + NTPs

Elongation

FIG. 5. Schematic model showing the stepwise formation of the transcription initiation complex at the mouse rDNA promoter. The order of assembly of the individual transcription factors and pol I as determined in this study is shown. Complex 5 is believed to represent a complete transcription initiation complex. The arrow indicates the transcription initiation site; the numbers at the top mark the nucleotide positions relative to the start site of transcription. The relative positions of the individual proteins and their structure are purely speculative and for illustration only. nd., not done.

plate and thus may be regarded as a commitment factor whose binding to the core promoter is the prerequisite for assembly of the basal transcription apparatus. Neither pol I, TIF-IA, or TIF-IC is required for the formation of this primary complex.

The question of whether or not TIF-IB can form a stable complex in the absence of UBF is an important one. There are two alternatives to explain the controversial results obtained in different laboratories showing that UBF is part of, or is required for, the assembly of complex 1. (i) TIF-IB is still contaminated by small amounts of UBF or (ii) there are species-specific differences in the cooperativity between UBF and TIF-IB-like factors. We strongly favor the latter alter- native for the following reasons. We have carefully analyzed the TIF-IB preparation used by a number of different methods (silver staining, footprinting analysis, rephosphorylation of phosphatase-treated factor preparation by casein kinase 11) and did not detect any UBF in the TIF-IB preparation. On the other hand, in a recent paper, McStay et al. (1991) have shown that in Xenopus laeuis the TIF-IB analogue Rib1 requires UBF for stable complex formation. The differences between this report and the data presented in this paper could be due to species-specific differences between the Xenopus and the mouse rDNA-binding properties. Both human and mouse UBF bind to the human rDNA promoter but only weakly interact with the mouse rDNA promoter. On the other hand, footprinting experiments with purified human and mouse SL1, respectively, demonstrated that human SL1 does not bind to its promoter in the absence of UBF, whereas mouse SL1 on its own recognizes the mouse promoter. Ap- parently, species-specific differences exist in the binding affinity of the TIF-IB analogues to their cognate promoter sequences, which in turn may result in different requirements for UBF in stable binding.

Although TIF-IB binds to the promoter by itself and forms a stable complex resistant to competition by a second promoter, the formation of productive complexes at the mouse promoter is enhanced by UBF. When UBF was present during the preincubation period, the number of productive complexes on the rDNA template was significantly increased (Fig. 2B). This finding is in accord with previous results demonstrating a strong cooperative complex between SL1 and UBF (Bell et al., 1988, 1990). Thus, one of the functions of UBF may be to recruit pol I and the other factors to the promoter by enhanc- ing or stabilizing TIF-IB binding.

Following binding of TIF-IB (and probably UBF) to the template, a higher order complex that includes pol I, TIF-IA, and TIF-IC is formed. This assembly appears to be the rate- limiting step in initiation and occurs in a highly ordered fashion. Using a protocol that combines template exclusion, order of addition, and inhibitor-resistance experiments, we have shown that in the next step, pol I is stably sequestered by complex 1 to generate complex 2. This complex exerts the same sensitivity to Sarkosyl as complex 1 and is transcriptionally inactive unless factors TIF-IC and TIF-IA have associated. Recent experiments from our laboratory have demonstrated that both TIF-IA and TIF-IC bind to pol I in solution (Schnapp et aL, 1990b).' Therefore, it is very likely that these two factors exclusively assemble into the complex via interaction with the polymerase. The association of the two pol I-binding factors with complex 2 is a strictly sequential process, too. First, TIF-IC is recruited, yielding complex 3. Binding of TIF-IC to the preinitiation complex, in turn, is a prerequisite for the final association of the growth-regulated factor TIF-IA. Addition of ATP and CTP (the first two nucleotides of mouse pre-rRNA) converts this final complex


(complex 4) into an initiated complex. The initiation phase, which is characterized by the formation of the first phosphodiester bond, gives rise to a transcription complex that is capable of elongation in the presence of high concentrations (0.3%) of Sarkosyl.

The identification of an ordered set of complexes, each containing some subset of the previously identified transcription factors, defines a reaction pathway for transcription initiation by pol I. Although the binding of each of these factors may not be strictly sequential in uiuo, with some binding together as pre-existing complexes, it is likely that the contacts between the factors defined by the ordered pathway shown in Fig. 5 occur in uiuo. Indeed, interactions of TIF- IC and TIF-IA, respectively, with pol I occur in the absence of template DNA (Schnapp et al., 1990b).2 The fact that in most groups working on pol I transcription, these two factors have not been identified and are not required for reconstitu- tion of transcriptional activity (Tower and Sollner-Webb, 1986; Bell et al., 1990; Smith et al., 1990), very likely reflects their failure to separate these activities from pol I by the fractionation procedure employed. Recent results from our laboratory suggest also that the DNA-binding factor UBF may interact with pol I. Using affinity chromatography with pol I immobilized on a column matrix, we found that UBF binds to and elutes at high salt concentration from this column.'j Therefore, it is conceivable that in the cell, most of the factors are associated with pol I as a multiprotein complex.

When we tested the energy requirement of rDNA transcription initiation by substituting ATP with AMP-PNP, we found that this analogue with a noncleavable P-y bond can be used to repalce ATP. Similar results have been reported by Gokal et al. (1990). This suggests that in the pol I system, ATP is not required as a source of energy and it is reasonable to conclude that neither initiation nor elongation by RNA polymerase I requires an ATPase activity. Similarly, ATP has been reported to be dispensable for pol I11 transcription initiation (Bunick et a i , 1982). In striking contrast, eukaryotic pol I1 transcription is dependent on a hydrolyzable P-y bond in ATP, which can also be supplied by dATP or araATP (Conaway and Conaway, 1988). ATPyS, on the other hand, has been shown to be a potent and reversible inhibitor of the ATP-requiring step in the initiation reaction of pol I1 transcripts (Bunick et al., 1982; Sawadogo and Roeder, 1984; Conaway and Conaway, 1988). The precise mechanism of this ATP dependence is not yet understood. It has been suggested that ATP may be used as a source of energy for some step in initiation, such as melting the DNA at the start site of transcription, or that ATP serves as a phosphate donor in a phosphorylation step. There is some experimental evidence for each of these hypotheses (Conaway and Conaway, 1989b; Arias et al., 1991). Concerning the pol I system, DNA melting occurs during initiation by Acanthumoeba pol I (Bateman and Psule, 1988), but no data concerning this phenomenon have been reported for the vertebrate RNA polymerase I. There- fore, we still don't know why for different classes of genes, fundamentally different mechanisms act at a step prior to synthesis of the first phosphodiester bond.

Acknowledgments-We thank W. Hadelt and S. Traub for help in extract preparation and fractionation and H. Rosenbauer and A. Kuhn for providing purified UBF.

REFERENCES

Arias, J. A., Peterson, S. R., and Dynan, W. S. (1991) J. Biol. Chem. 266,8055-8061

' G. Heilgenthal, unpublished data.

Bateman, E., and Paule, M. R. (1988) Mol. Cell. Bid. 8, 1940-1946 Bell, S. P., Learned, R. M., Jantzen, H.-M., and Tjian, R. (1988)

Bell, S. P., Jantzen, H.-M., and Tjian, R. (1990) Genes & Deu. 4,

Bogenhagen, D. F., Wormington, W. M., and Brown, D. D. (1982)

Bunick, D., Zandomeni, R., Ackermann, S., and Weinmann, R. (1982)

Buratowski, S., Hahn, S., Guerente, L., and Sharp, P. A. (1989) Cell

Buttgereit, D., Plugfelder, G., and Grummt, I. (1985) Nucleic Acids

Cizewski, V., and Sollner-Webb, B. (1983) Nucleic Acids Res. 11,

Clos, J., Buttgereit, D., and Grummt, I. (1986a) Proc. Natl. Acad. Sci.

Clos, J., Normann, A., Ohrlein, A., and Grummt, I. (1986b) Nucleic

Conaway, R. C., and Conaway, J. W. (1988) J. Biol. Chem. 263,

Conaway, J. W., and Conaway, R. C. (1989a) J. Biol. Chem. 264,

Conaway, R. C., and Conaway, J . W. (1989b) Proc. Natl. Acad. Sci.

Conaway, R. C., and Conaway, J. W. (1990) J. Biol. Chem. 265,

Conaway, J. W., Reines, D., and Conaway, R. C. (1990) J. Biol. Chem.

Dignam, J. D., Lobowitz, R. M., and Roeder, R. G. (1983) Nucleic

Flores, O., Maldonado, E., Burton, Z., Greenblatt, J., and Reinberg,

Flores, O., Maldonado, E., and Reinberg, D. (1989) J. Biol. Chem.

Gokal, P. K., Mahajan, P. B., and Thompson, E. A. (1990) J. Biol.

Grummt, I. (1981) Proc. Natl. Acad. Sci. CJ. S. A . 78, 727-731 Grummt, I. (1989) in Nucleic Acids and Molecular Biology (Eckstein,

F., and Lilley, D. M. J., eds) Vol. 3, pp. 148-163, Springer-Verlag, Berlin

Science 241, 1192-1197

943-954

Cell 28,413-421

Cell 29,877-888

56,549-561

Res. 13,8165-8179

7043-7056

U. S. A. 83,604-608 ,,

Acids Res. 14, 7581-7595

2962-2968

2357-2362

U. S. A. 86, 7356-7360

7559-7563

265,7552-7558

Acids Res. 11,1475-1489

D. (1988) J. Biol. Chem. 263,10821-10816

264,8913-8921

Chem. 265,16234-16243

Grummt, I., Roth, E., and Paule, M. (1982) Nature 296,173-174 Hawley, D. K., and Roeder, R. G. (1985) J. Biol. Chem. 260, 8163-

Inostroza, J., Flores, O., and Reinberg, D. (1991) J. Biol. Chem. 266,

Kato, H., Nagamine, M., Kominami, R., and Muramatsu, M. (1986)

Learned, R. M., Cordes, S., and Tjian, R. T. (1985) Mol. Cell. Biol. 5,

Learned, R. M., Learned, T. K., Haltiner, M. M., and Tjian, R. T.

Mahaian. P. B.. and ThomDson, A. E. (1990) J. Biol. Chem. 265,

8172

9304-9308

Mol. Cell. Biol. 6, 3418-3427

1358-1369

(1986) Cell 45,847-857

16225-16233

Mol. Cell. Biol. 10, 6335-6347

~.

Maldonado, E., Ha, I., Cortes, P., Weis, L., and Reinberg, D. (1990)

McStay, B., Hu, C. H., Pikaard, C. S., and Reeder, R. H. (1991)

Mishima, Y . , Finanscek, I., Kominami, R., and Muramatsu, M. (1982)

Nakajima, N., Horikoshi, M., and Roeder, R. G . (1988) Mol. Cell.

Onishi. T.. Berdund. C.. and Reeder. R. H. (1984) Proc. Natl. Acad.

EMBO J. 10,2297-2303

Nucleic Acids Res. 10, 6659-6670

Biol. 8,4028-4040

Sci. U . S . A. 81, 484-487 Palmer, J. M., and Folk, W. R. (1990) Trends Biochem. Sci. 15,300- 304

3321 Reinberg, D., and Roeder, R. G. (1987) J. Biol. Chem. 262, 3310-

Reinberg, D., Horikoshi, M., and Roeder, R. G. (1987) J. Btol. Chem.

Sawadogo, M., and Roeder, R. G. (1984) J. Biol. Chem. 259, 5321-

Sawadogo, M., and Sentenac, A. (1990) Annu. Reu. Biochem. 59,

Schnapp, A., Clos, J., Hadelt, W., Schreck, R., Cvekl, A,, and Grummt,

Schnapp, A., Pfleiderer, C., Rosenbauer, H., and Grummt, I. (1990b)

262,3322-3330

5326

711-754

I. (1990a) Nucleic Acids Res. 18, 1385-1393

EMBO J. 9, 2857-2863


Schnapp, A,, Rosenbauer, H., and Grummt, I. (1991) Mol. Cell. Biochem. lo?., 137-147

Skinner, J. A., Ohrlein, A., and Grummt, I. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 2137-2141

Smith, S. D., Oriahi, E., Lowe, D., Yang-Yeng, H.-F., O’Mahony, D., Rose, K., and Rothblum, L. I. (1990) Mol. Cell. Biol. 10, 3105-3116

Tanaka, N., Kato, H., Ishikawa, Y., Hisatake, K., Tashiro, K., Kom- inami, R., and Muramatsu, M. (1990) J. Bid. Chem. 265, 13836- 13842

Tower, J., and Sollner-Webb, B. (1987) Cell 50, 873-883 Tower, J., Culotta, V., and Sollner-Webb, B. (1986) Mol. Cell. Biol.

Van Dyke, M. W., Sawadogo, M., and Roeder, R. G. (1989) Mol. Cell.

Wandelt, C., and Grummt, I. (1983) Nucleic Acids Res. 11, 3795-

Weil, P. A., Luse, D. S., Segall, J., and Roeder, R. G. (1979) Cell 18,

6,3451-3462

Biol. 9, 342-344

3809

469-484

THE OF CHEMISTRY Vol. 266, No. 36, Issue of 25, pp. 24588 ...THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc Vol. 266,

Documents