THE JOURNAL OF BIOLOGICAL CHEMISTRY

Prrnted in U. S. A. Vol. 255, No. 24, Issue of December 25, pp. 11986-11991, 1980

Multiple Factors Are Required for the Accurate Transcription of Purified Genes by RNA Polymerase III*

(Received for publication, June 19, 1980, and in revised form, August 15, 1980)

Jacqueline SegallS, Takashi Matsuig, and Robert G . Roederfl From the Departments of Biological Chemistry and Genetics, Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, Missouri 63110

Cell-free extracts (5-100) prepared from cultured mammalian KB cells have previously been shown to direct accurate and selective transcription of class 111 genes by RNA polymerase III. We have fractionated the KB S-100 and have found that multiple components are essential for the accurate transcription of these genes. After the S-100 has been separated into four different protein fractions by chromatography on phos- phocellulose, two fractions are required, in addition to RNA polymerase III, for active and selective transcrip- tion of the virus-associated RNA1 gene of adenovirus 2 and a tRNA gene; a third fraction is required, along with these components, for the reconstitution of 5 S RNA gene transcription. At least two of these compo- nents are distinct from the four factors required for accurate initiation of transcription by RNA polymerase I1 (Matsui, T., Segall, J., Weil, P. A., and Roeder, R. G. (1980) J. BioL Chem 255,11992-11996).

Soluble cell-free extracts have recently been shown to direct the accurate transcription of eukaryotic genes in purified DNA templates. Extracts prepared from a variety of sources including cultured human cells (1-5), mature Xenopus oocytes (6, 7), and Xenopus germinal vesicles (8-14) support accurate transcription by the endogenous RNA polymerase I11 of sev- eral class 111 genes, including cloned 5 S RNA and tRNA genes and the virus-associated RNA genes of adenovirus 2. In addition, a mammalian cell-free extract has been shown to direct accurate transcription initiation by an exogenous RNA polymerase I1 at the major late promoter of adenovirus (15) and at the mouse major P-globin gene promoter (16). Since the corresponding purified RNA polymerases transcribe pu- rified class I1 and 111 genes randomly, these crude extracts must contain factors which do not copurify with the enzymes but which are essential for accurate transcription.

Transcription of in vitro mutated genes in these crude extracts has allowed the deduction of DNA sequences critical for the proper transcription of 5 S RNA and tRNA genes (17-

* These studies were supported by Research Grants CAI6640 and CA23615 from the National Cancer Institute and Research Grant NP284 from the American Cancer Society. Cell culture media, KB cells, and virus were prepared in a Cancer Center facility funded by Cancer Support Grant P30 CA 176217 from the National Cancer Institute to Washington University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by a feilowship from the Damon Runyon-Walter Win- chell Cancer Fund. Present address, Department of Biochemistry, University of Toronto, Faculty of Medicine, Toronto, Ontario, Canada M5S 1A8.

8 Present address, Department of Molecular Biology, School of Medicine, Yahatanishi-Kn, Kitakyushu, Japan.

7 Camille and Henry Dreyfus Teacher-Scholar Awardee.

20). However, a detailed understanding of the nature and mechanism of action of the various factors present in these extracts requires that they be isolated and purified. Initial fractionation of soluble extracts from Xenopus ovaries and from cultured mammalian cells has indicated that at least two components are required, in addition to RNA polymerase 111, for the accurate transcription of purified 5 S RNA genes (3, 7). Moreover, one of these factors has been purified from Xenopus ovaries and has been shown to bind to a previously described internal control region of 5 S RNA genes (7, 17, 18). These studies have indicated the ultimate feasibility of study- ing selective eukaryotic transcription in an in vitro system reconstituted from purified components.

In this report, we have extended the fractionation of the soluble extract prepared from human cells and our initial studies have indicated that multiple components are necessary for the accurate transcription of class 111 genes. After an initial chromatographic procedure separating the extract into four fractions, we find that two fractions ( i n addition to RNA polymerase 111) are required for active transcription of a tRNA gene and the VA’ RNA1 gene of Ad2. In addition to these two fractions, a third fraction is required for the recon- stitution of 5 S RNA gene transcription. The relationship of these factors to the several factors involved in class I1 gene transcription (accompanying paper, Ref. 21) is discussed.

EXPERIMENTAL PROCEDURES

Cell Culture, Preparation of Cell Extracts, and DNA Purifica- tion-Procedures for cell culture, the preparation of the KB S-100 extract, and Ad2 and plasmid DNA purification have been described previously (2). After preparation, the S-100 extract was dialyzed for 6 to 8 h against Buffer A (20 mM Hepes (pH 7.9), 20% glycerol, 0.2 mM EDTA, and 0.5 m dithiothreitol) containing 0. I M KC1 and then stored at -80°C after a 10-min centrifugation at 12,000 X g. The plasmid pXbsl was obtained from J. L. Doering and contains one Xenopus borealis somatic 5 S DNA repeat (gene plus spacer) inserted into pMB9 (22, 23). The plasmid pXltmetl was constructed by A. Lassar and contains a single Xenopus laevis tRNA? gene (the largest Msp I fragment of At210 (24)) inserted into pBR322.

Chromatography of the KB S-100-A soluble extract (S-100) (12 to 16 mg of protein/ml of S-100; 1.2 X 10’ cell equivalent/ml of S-100) in Buffer A containing 0.1 M KC1 (see above) was applied to a phosphocellulose (Whatman P11) column equilibrated with the same buffer (10 mg of protein/ml of bed volume). The column was washed with this buffer and the bound protein was then sequentially step- eluted with Buffer A containing 0.35, 0.6, and 1.0 M KC1. This last buffer also contained 0.2 mg/ml of bovine serum albumin (Pentex). For this and subsequent column chromatography, fractions equivalent to 10% of the bed volume were collected and the appropriate break- through and step-eluted fractions (2 to 4 fractions) were pooled on the basis of their absorbance at 280 nm. Pooled fractions which were not used for subsequent chromatography were dialyzed against Buffer A containing 5 m MgClz (Buffer B) and 0.1 M KC1 for 4 to 10 h and then stored in aliquots at -80°C. The combined phosphocellulose 0.6

’ The abbreviations used are: VA, virus-associated; Ad2, adenovirus 2; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

11986

RNA Polymerase 111 Transcription Factors 11987

M KC1 step fractions were adjusted to 5 mM MgClz and dialyzed against Buffer B to a final concentration of 0.1 M KCI. After a low speed centrifugation, this pool was loaded onto a column of DEAE- cellulose (Whatman DE52) equilibrated with Buffer B containing 0.1 M KC1 (3 mg of protein/ml of bed volume). The column was washed with this buffer, bovine serum albumin was added to 0.2 mg/ml to the breakthrough fractions, and the bound protein was then step- eluted with Buffer B containing 0.25 M KCI.

Enzyme Preparation-The accompanying paper (21) describes the preparation of KB RNA polymerase I1 from the nuclear pellets (P- 100) recovered in the preparation of the S-100s. The DEAE-cellulose breakthrough fraction from this procedure (containing RNA polym- erase I and 111 activities) was used to prepare RNA polymerase 111. This fraction was applied to a column of DEAE-Sephadex equili- brated with a buffer containing 50 mM Tris-HC1 (pH 7.9), 25% glycerol, 0.1 M EDTA, 0.5 mM dithiothreitol, and 115 mM (NH4)SOd. The column was washed with this buffer and then with buffer containing 160 m~ (NH4)zS04. RNA polymerase I11 was then step-eluted from the column with buffer containing 500 m~ (NH&SO4. The enzyme was then concentrated by chromatography on phosphocellulose.

Transcription Assays-The standard 50-pl transcription reaction contained 12 mM Hepes (pH 7.9), 70 mM KCI, 6 mM MgCIz, 12% glycerol, 0.3 mM dithiothreitol, 25 p~ [a-32P]GTP (1.6 Ci/mM), 600 p~ each of ATP, CTP, and UTP, and either 18 pg/ml of Ad2 DNA or 9 pg/ml of pXbsl or pXltmetl DNA. Where indicated, the reactions were supplemented with 50 to 100 units (as defined in Ref. 2) of KB RNA polymerase 111. The reactions were incubated and the RNAs were purified and fractionated by electrophoresis on 12% polyacryl- amide slab gels as described in Weil et al. (15).

RESULTS

Fractionation of Class 111 Gene Transcription Factors-A soluble extract (S-100) prepared from cultured human KB cells was separated into four distinct protein fractions by chromatography on phosphocellulose as follows. The extract was loaded onto a column of this ion exchanger in a buffer containing 0.1 M KC1 and the bound proteins were then sequentially eluted with buffers containing 0.35, 0.6, and 1.0 M KCl; panel A of Fig. 1 shows the distribution of protein between the four fractions obtained in this separation. The individual fractions were assayed for their ability to direct accurate transcription by RNA polymerase I11 purified from KB cells. In order to detect any gene-specific transcription factors, we have simultaneously assayed for the transcription of the VA RNA1 gene on purified Ad2 DNA, the X. borealis somatic 5 S gene of the plasmid pXbsl (22, 23) and the X. laeuis tRNA;”” gene of the plasmid pXltmetl (see under “Experimental Procedures”). In the presence of the unfrac- tionated S-100, the major RNA synthesized by RNA polym- erase I11 on Ad2 DNA, as visualized by electrophoretic anal- ysis, is VA RNA1 (Fig. lB, lane 1 ) (1, 2, 5) and on pXbsl the major transcript is 5 S RNA (Fig. lD, lane 1) . Transcription of pXltmet, in the presence of the S-100 results in a discrete- sized RNA which we presume on the basis of its size to be a precursor to the mature tRNA transcript (see below) (Fig. IC, lane 1) .

The electrophoretic analysis of the RNAs synthesized by RNA polymerase I11 in the presence of the various phospho- cellulose fractions is also shown in Fig. 1. None of the individ- ual fractions alone actively supported selective transcription of the VA RNA1 gene of Ad2 (Fig. lB, lanes 2 to 5), although the 0.6 M KC1 step fraction did lead to a very low level of VA RNA1 synthesis (Fig. lB, lane 4). However, a high level of VA RNAr gene transcription was obtained when transcription was carried out in the presence of a combination of the 0.35 M KC1 and 0.6 M KC1 step fractions (Fig. lB, lane 6). Similarly, no single fraction could actively direct selective transcription of the tRNA gene or 5 S RNA gene templates (Fig. 1, C and D, lanes 2 to 5). However, as for VA RNA1 gene transcription, the 0.6 M KC1 step fraction led to a very low level of transcrip- tion of the tRNA gene (Fig. lC, lane 4) and a combination of

the 0.35 M KC1 and 0.6 M KC1 step fractions supported a high level of transcription of this gene (Fig. IC, lane 6). In contrast, neither this combination nor any other painvise combination of fractions supported selective transcription of the 5 S RNA gene template (Fig. lD, lanes 6, 8, 9, and data not shown). Accurate transcription of this template required the presence of the breakthrough fraction in addition to the 0.35 M KC1 and the 0.6 M KC1 step fractions (Fig. lD, lane 7). Hence, reconstitution of accurate transcription after an initial chro- matographic separation of the S-100 into four fractions indi- cates that at least two components are required for VA RNA1 and tRNA gene transcription and that at least three compo- nents are required for 5 S gene transcription.

Accurate Transcription Dependent on Exogenous RNA Polymerase 111-The cell-free extract contains a significant amount of RNA polymerase I11 (2) which, after the phospho- cellulose fractionation, is recovered in both the 0.35 M KC1 and the 0.6 M KC1 step fractions. Hence, it is possible that one of the transcription factors could in fact be RNA polymerase 111. However, all of the transcription reactions described above

0

Frootlu No.

C o o a D n o o a b c d c c

b b b c

a a a

r l o o a b c d c c c b b b

I 2 3 4 5 6 7 8 9 -” c

b b b a o a

rmoo b c d c c c I 2 3 4 5 6 7 8 9 l 2 3 4 5 6 ? 8 9

“0

5s- c w

FIG. 1. Separation of class III gene transcription compo- nents on phosphocellulose. A soluble extract (S-100) prepared from KB cells was chromatographed on phosphocellulose as described under “Experimental Procedures” to give a breakthrough fraction (a) and 0.35 M KC1 ( b ) , 0.6 M KC1 (c), and 1.0 M KC1 (d) step-eluted fractions. The absorbance profile (Am ,,,,,) of this fractionation scheme is shown in A. The fractions were assayed for their ability to direct accurate transcription on Ad2 DNA (B) , pXltmet, DNA (0, or pXbsl DNA (D). The autoradiograms show the gel analyses of RNAs synthesized in the presence of the S-100 (lane I ) ; the breakthrough fraction (lane 2); the 0.35 M KC1 step fraction (lane 3); the 0.6 M KC1 step fraction (lane 4); the 1.0 M KC1 step fraction (lane 5) a combi- nation of the 0.35 M KC1 and 0.6 M KC1 step fractions (lane 6); a combination of the breakthrough fraction and the 0.35 M KC1 and 0.6 M KC1 step fractions (lane 7); a combination of the breakthrough fraction and the 0.35 M KC1 step fraction (lane 8); or a combination of the breakthrough fraction and the 0.6 M KC1 step fraction (lane 9). Ten microliters of each fraction was used. All reactions contained 50 units of KB RNA polymerase 111. The positions of VA RNA,, 5 S RNA, and the tRNAP’‘ precursor (ptmet) are indicated. The exposure time for the autoradiograms of B and D was three times that of C.

11988 RNA Polymerase 111 Transcription Factors

were carried out in the presence of exogenous (purified) RNA polymerase 111. Although this purified enz-yme can stimulate selective transcription in the reconstituted transcription re- action containing the phosphocellulose 0.35 M KC1 and 0.6 M KC1 step fractions (Fig. 2, lanes I and 2), it does not lead to selective transcription in the presence of either fraction alone (Fig. lB, lanes 3 and 4). This implies that each of these fractions contains a transcription factor(s) distinct from RNA polymerase 111. To ascertain that exogenous RNA polymerase I11 can substitute for the endogenous enzyme, the latter enzyme was selectively removed from the 0.35 M KC1 and 0.6 M KC1 phosphocellulose step fractions (see legend to Fig. 2). As anticipated, there is no RNA synthesis when a combination of these latter RNA polymerase-free phosphocellulose frac- tions (and also the breakthrough fraction for the 5 S RNA gene template) are used in the reconstituted transcription reactions (Fig. 2, lanes 5 to 7). If the reactions are supple- mented with purified RNA polymerase 111, then active and accurate transcription of the class 111 genes is restored (com- pare lanes 8 to 10 and 5 to ?of Fig. 2 ) . Hence, purified enzyme can substitute for the endogenous activity indicating that at

a b b b b b' b' E' b' b' b'

a

- c c c c c' C' c' C' c' c' I 2 3 4 a

- + + +

5 6 7 8 9 1 0

" - + + + polllr FIG. 2. Reconstituted transcription dependent on exogenous

RNA polymerase 111. A phosphocellulose 0.35 M KC1 step fraction ( 6 ) and 0.6 M KC1 step fraction (c) similar to those of Fig. 1 were dialyzed into Buffer A (see "Experimental Procedures") containing 130 and 155 mM ammonium sulfate, respectively, and passed through columns of DEAE-Sephadex equilibrated with the same buffers. The DEAE-Sephadex breakthrough fractions (6' and c' from the phos- phocellulose 0.35 M KC1 and 0.6 M KC1 step fractions, respectively) did not contain any RNA polymerase 111 activity. The autoradiogram shows the gel analyses of RNAs synthesized on Ad2 DNA (lanes 1,2, 5, and 8). pXltmetl DNA (lanes 3, 6. and 9), or pXbsl DNA (lunes 4, 7, and 10) in the presence of a mixture of 10 pl each of the phosphocellulose 0.35 M KC1 and 0.6 M KC1 step fractions ( b + c) (lunes I to 4) or in the presence of a mixture of 10 p1 each of the DEAE-Sephadex breakthrough fractions obtained from the phospho- cellulose 0.35 M KC1 and 0.6 M KC1 step fractions (b' + c ' ) (lanes 5 to 10). The reactions of lanes 4, 7, and 10 also contained 10 pl of the phosphocellulose breakthrough fraction ( a ) . The reactions of lanes 2, 3, 4, 8, 9, and 10 were supplemented with 50 units of KB RNA polymerase 111 as denoted by the +. (As seen in lanes 4 and 10, transcription of pXbsl has, in this case, resulted in the synthesis of two RNAs differing in size by only a few nucleotides. We have not investigated the nature of these transcripts but presume that they result from termination a t different sites (see Ref. 2)).

I

1 2 3 4 5 6 7 8 9 1 0 1 1

FIG. 3. Processing of the tRNA?" precursor. a"2P-tRNAP'" precursor transcript (ptmet) was purified from a standard transcrip- tion reaction carried out in the presence of an S-100. The autoradi- ogram shows an analysis of this RNA incubated for increasing lengths of time in the presence of 15 pI of the phosphocellulose 0.35 M KC1 step fraction (see Fig. 1). These reactions were equivalent to the standard transcription reaction except for the omission of [a-'"P]GTP and a DNA template. [a-:"P]VA RNA1 was also included in the reactions of lanes I and 2. The reaction of lane 10 also contained 15 pl of the phosphocellulose breakthrough fraction. The reactions were incubated at 30°C for 0 min (lanes 1, 3, and ZZ), 5 min (lane 4 ) , 10 rnin (lane 5). 20 min (lane 6) . 40 min (lane 7). 60 min (lane 8). or 80 min (lanes 2, 9, and 10). The dotted line indicates the position of the xylene cyano1 FF dye front.

least two factors other than RNA polymerase 111, which are required for active and accurate transcription of class I11 genes, bind to phosphocellulose.

Processing of a Precursor tRNA Transcript-As shown above, transcription of pXltmet, in the presence of the S-100 extract gives rise to a single RNA species (ptmet) (Fig. IC, lane I) which we presume on the basis of its size to be the precursor to the mature tRNA?" species. This transcript is similar in size to a transcript of this gene synthesized in the presence of a Xenopus ovary extract and shown to be a precursor to the mature tRNA transcript (see Ref: 7). In the reconstituted transcription reaction containing the phospho- cellulose 0.35 M KC1 and 0.6 M KC1 fraction, RNAs of approx- imately the size of the mature tRNA species can be seen, suggesting that some processing of the precursor is occurring (Fig. IC, lane 6). To investigate this possibility, the precursor tRNA? species was purified from a transcription reaction carried out in the presence of the S-100.and then incubated for increasing lengths of time in, the presence of the phospho- cellulose 0.35 M KC1 step fraction (Fig. 3). This resulted in the precursor transcript being reduced to the approximate size of the mature tRNA within 40 min with no further major change over an additional 40 min of incubation. In contrast, VA RNA, was unaffected by incubation in the presence of the phospho- cellulose 0.35 M KC1 step fraction (Fig. 3, lanes I and 2). If the phosphocellulose breakthrough fraction was added io the 0.35 M KC1 step fraction, processing of the precursor tRNA was reduced (Fig. 3, lane 10 and Fig. IC, lane 7). This is consistent with the lack of processing observed when transcription of pXltmet, is carried out in the presence of the S-100 and suggests that an inhibitory activity(ies) must be removed in order for processing to occur. A similar investigation of the 0.6 M KC1 step fraction indicates that this fraction contains both a processing activity and also a component that inhibits processing (data not shown).

We have not determined whether the sites at which the

RNA Polymerase 111

precursor tRNA is cleaved in vitro are the same sites at which processing occurs in vivo. Hence, we cannot exclude the possibility that the in vitro processing observed simply reflects trimming of the exposed precursor segments at nonspecific sites leaving a resistant tRNA core. Relevant to this possibility is the observation that KB cells contain an endonuclease (RNase NU) which will cleave RNAs that are unstable in vivo but will not cleave RNAs which are stable in vivo (25). We have not further tested this possibility.

Unique Class ZZZ and Class ZZ Gene Transcription Fac- tors-The KB S-100 has also been shown to direct the accu- rate initiation of transcription by RNA polymerase I1 at the major late promoter of Ad2 (15) and at the mouse major p- globin promoter (16). The accompanying paper (21) shows that when an S-100 is fractionated on phosphocellulose as described here, a combination of the breakthrough fraction and the 0.6 M KC1 and the 1.0 M KC1 step fractions is necessary to effect maximal transcription from the major late promoter of Ad2. As summarized in Fig. 4, both the phosphocellulose breakthrough fraction and 0.6 M KC1 step fraction contain a component(s) necessary for class I1 and class 111 ‘:ene tran- scription. This raises the possibility that a common factor could be involved in the transcription of both clae ,es of genes. The most likely mechanism of action of a transcription factor would be through an interaction with either RNA polymerase or a DNA control sequence; however, since the structures of RNA polymerase I1 and I11 are quite distinct (26) and since

b l : KB S-100

PI1 (0.1 M KCI)

a BT 0.35 0.6 Lo M KC1

IUA IIIB IIIC I IA

, ‘ B ~ ~ E 5 2 ~ O ~ M KC0

e BT 0.25 M KC1

lI B,C E C

, ~ 1 D; cell;se (0.1 M KCI)

9 BT 0.3 0.6 1.0M KC1

IIB I C FIG. 4. Separation of RNA polymerase I1 and 111 transcrip-

tion factors. The chromatographic steps are described under “Ex- perimental Procedures’’ of this and the accompanying paper (21). Transcription factors are denoted IZZA, I IA, etc., for convenience only and refer to chromatographic fractions which may contain several transcription components. I I IA , I I IB , and IZZC refer to factors re- quired for the accurate transcription of class 111 genes. The factors ZIZB and IIIC are required for the selective transcription of VA RNA and tHNA genes and the factors I I IA , ZIIB and IIIC are required for selective 5 S RNA gene transcription. Accurate initiation of transcrip- tion at the major late promoter of Ad2 requires the factors ZZA, IZB, ZIC, and IID (21). BT, P11, and DE52 denote breakthrough, phos- phocellulose, and DEAE-cellulose, respectively. The KC1 concentra- tions indicate either the salt concentration at which protein was loaded onto or eluted from a column.

Transcription Factors 11989

A

s 100 I PI1

ET a

C

b b b b c e f e f . ” ” _

035 66 IlOMKCI VA, b c d

“L DE52 (OM KCI)

ET 0.25M KC1 e f

b b b b c e f e f

I 2 3 4 5 6 I) C .

D a a a a b b b b

c e f e f 1 2 3 4 5 6

t t

FIG. 5. DEAE-cellulose chromatography of the phosphocel- lulose 0.6 M KC1 step fraction. The phosphocellulose 0.6 M KC1 fraction of Fig. 1 was chromatographed on DEAE-cellulose (DE52) as described under “Experimental Procedures” to give a breakthrough fraction ( e ) and a 0.25 M KC1 step-eluted fraction (f) (A). The autoradiograms show the gel analyses of RNAs synthesized on Ad2 DNA (B) , pXltmet, DNA (0, or pXbsl DNA (D) in the presence of the 0.35 M KC1 phosphocellulose step fraction alone (lane I ) or this fraction mixed with either the 0.6 M KC1 phosphocellulose step fraction (lane 2). the DEAE-cellulose breakthrough fraction (lane 3). or the DEAE-cellulose step fraction (lane 4). The reaction of lane 5 contained the DEAE-cellulose breakthrough fraction alone and the reaction of lane 6 contained the DEAE-cellulose step fraction alone. The reactions of lanes 1 to 4 of D also contained the phosphocellulose breakthrough fraction. Ten microliters of each fraction was used. All reactions were supplemented with 50 units of KB RNA polymerase 111.

no putative regulatory sequence common to both class I1 and class I11 genes has been identified, a common transcription factor seems unlikely. Moreover, the class I11 gene transcrip- tion factor present in the phosphocellulose breakthrough frac- tion is not a general class I11 gene transcription factor: it is required for 5 S RNA gene transcription but not for VA RNA, or tRNA gene transcription. This suggests that this factor acts by recognizing a unique region of 5 S DNA and would not also be involved in class I1 gene transcription.

To determine whether the class I11 and class I1 gene tran- scription components present in the phosphocellulose 0.6 M KC1 step fraction are distinct, this fraction was chromato- graphed on DEAE-cellulose to give a breakthrough fraction

11990 RNA Polymerase III Transcription Factors

(0.1 M KCl) and a single step-eluted fraction (0.25 M KCl). The DEAE-cellulose breakthrough fraction cannot substitute for the input phosphocellulose 0.6 M KC1 step fraction in directing active transcription of any of the class 111 genes (Fig. 5, B, C, and D, lane 3). However, the DEAE-cellulose 0.25 M KC1 step fraction will reconstitute with the phosphocellulose 0.35 M KC1 step fraction to direct accurate transcription ofthe VA RNA1 gene (Fig. 5B, lane 4) and the tRNA? gene @anel C, lane 4); and with the phosphocellulose breakthrough frac- tion and 0.35 M KC1 step fraction to direct accurate transcrip- tion of the 5 S RNA gene (panel D, lane 4). In contrast, as demonstrated in the accompanying paper (21), it is the DEAE- cellulose breakthrough fraction that contains a transcription factor(s) required for active transcription from the major late promoter of Ad2 by RNA polymerase I1 (Fig. 4). Hence, the class I11 and class I1 transcription factors present in the phosphocellulose 0.6 M KC1 step fraction are different. Also, it can be noted that when the DEAE-cellulose 0.25 M KC1 step fraction substitutes for the phosphocellulose 0.6 M KC1 step fraction in transcription of pXltmetl DNA (in the presence of the phosphocellulose 0.35 M KC1 step fraction) processing of the precursor tRNA is much more efficient (Fig. 5C, lanes 2 and 4), again suggesting the removal of an inhibitory activity (see above).

DISCUSSION

In this and the accompanying report (21), we have demon- strated that multiple factors are involved in directing the accurate in vitro transcription of purified DNA templates by RNA polymerases I1 and 111. This has been shown by the analysis of RNAs synthesized on various templates in the presence of chromatographic fractions derived from a KB S- 100. The general fractionation scheme is shown in Fig. 4. In the case of the VA RNA1 and tRNA genes, transcription requires, in addition to RNA polymerase 111, two distinct fractions (the phosphocellulose 0.35 M KC1 and 0.6 M KC1 step fractions). We do not yet know whether both fractions contain components absolutely required for selective transcription or whether the component(s) of one fraction simply stimulates a low level of accurate transcription mediated by a compo- nent(s) in the other fraction. This latter possibility is suggested by the trace amount of selective VA RNA1 and tRNA gene transcription which is observed in the presence of the phos- phocellulose 0.6 M KC1 step fraction alone but which is greatly stimulated by the addition of the 0.35 M KC1 step fraction (Fig. 1, B and C, lanes 4 and 6). However, this result could also be due simply to a slight contamination of the higher salt- eluted fraction with a selectivity component which elutes mainly in the previous step. In the case of the 5 S RNA gene, selective transcription requires a third fraction in addition to the two fractions necessary for VA RNA1 and tRNA gene transcription.

In addition to providing information on general transcrip- tion factors, these chromatographic studies have indicated the presence in the crude extract of an activity(ies) which may mediate the processing of a tRNA precursor. More detailed analyses of tRNA processing in Xenopus oocyte-derived ex- tracts have been presented (9-14) and it has been shown that the processing activity is separable from the transcription factors (12).

The studies described here give only an indication of the minimal number of factors involved in selective transcription since any of the relatively crude fractions required in the reconstituted reaction could contain more than one compo- nent. In fact, preliminary fractionation of the proteins in the phosphocellulose 0.6 M KC1 step fraction has indicated the presence of a component necessary for the transcription of all

three genes (VA RNAI, tRNA, and 5 S RNA) and also a component uniquely required for VA RNAI gene transcrip- tion.2 Thus, it appears that there may be both gene-specific as well as generd class 111 gene transcription factors. A clear definition of the multiplicity and specificity of these factors awaits their complete purificaton. We do know, however, that most, or all, of the class I11 factors identified thus far are distinct from those factors (at least four) which are required for the accurate initiation of transcription at a class 11 gene promoter (see under “Results,” Fig. 4, and the accompanying paper, Ref. 21). The existence of such a multiplicity of tran- scription factors which are required for accurate gene tran- scription, but do not by themselves regulate gene expression (see below), indicates the complex nature of eukaryotic tran- scription.

As discussed previously (2-4, 15, 16), the existing cell-free transcription systems fail to reproduce the in vivo pattern of gene regulation. Thus, the VA RNA genes (expressed late in the Ad2 lytic cycle) are accurately transcribed in extracts prepared from uninfected KB cells; the oocyte type 5 S genes are accurately transcribed in extracts prepared from cells which contain, but do not express these genes (2). Hence, it appears that the transcription factors detected in the extracts thus far, although necessary and sufficient for the accurate transcription of purified genes, are, by themselves, insufficient for the regulation of gene expression. The purification and analysis of the factors described here should lead to an un- derstanding of their sites and mechanism of action and, hope- fully, provide a basis for the identification and study of tran- scription regulatory factors.

Acknowledgments-We thank Tony Weil for valuable contribu- tions to this study, Andrew Lassar, Jeffrey Doering, and Stuart Clarkson for recombinant DNA plasmids, and David Lee for a critical reading of the manuscript.

REFERENCES 1. Wu, G. J. (1978) Proc. Natl. Acad. Sci. U. S. A. 75,2175-2179 2. Weil, P. A., Segall, J., Harris, B., Ng, S.-Y., and Roeder, R. G.

(1979) J. Biol. Chem. 254,6163-6173 3. Roeder, R. G., Engelke, D. R., Harris, B., Ng, S.-Y., Segall, J.,

Shastry, B. S., and Weil, P. A. (1979) in Eukaryotic Gene Regulation (Axel, R., Maniatis, T., and Fox, C. F., eds) pp. 521- 540, Academic Press, New York

4. Roeder, R. G., Engelke, D. R., Luse, D. S., Ng, S.-Y., Segxll, J., Shastry, B. S., and Weil, P. A. (1979) in RNA Polymerase, Transfer RNA, and Ribosomes: Their Genetics and Evolution (Osawa, S., ed) University of Tokyo Press, Tokyo, in press

5. Wu, G. (1980) J. Biol. Chem. 255, 251-258 6. Ng, S.-Y., Parker, C. S., and -Roeder, R. G. (1979) Proc. Natl.

7. Engelke, D. R., Ng, S.-Y., Shastry, B. S., and Roeder, R. G. (1980)

8. Birkenmeyer, E. H., Brown, D. D., and Jordan, E. (1978) Cell 15,

9. Schmidt, O., Mao, J. I., Silverman, S., Hoveman, S., and Sok D.

Acad. Sci. U. S. A. 76, 136-140

Cell 19, 717-728

1077-1086

(1978) Proc. Natl. Acad. Sei. U . S. A. 75,4819-4823 10. Garber, R. L., and Gage, L. P. (1979) Cell 18,817-828 11. Hagenbuchle, O., Larson, D., Hall, G. I., and Sprague, K. U. (1979)

Cell 18, 1217-1229 12. Mattoccia, E., Baldi, M. I., Carrara, G., Fruscoloni, P., Benedetti,

P., and Tocchini-Valentini, G. P. (1979) Cell 18,643-648 13. Ogden, R. C., Beckman, J. S., Abelson, J., Kang, H. S., Soll, D.,

and Schmidt, 0. (1979) Cell 17, 399-406 14. Silverman, S., Schmidt, O., SOU, D., and Hovemann, B. (1979) J.

Biol. Chem. 254, 10290-10294 15. Weil, P. A., Luse, D. S., Segall, J., and Roeder, R. G. (1979) Cell

16. Luse, D. S., and Roeder, R. G. (1980) Cell 20,691-700 17. Sakonju, S., Bogenhagen, D. F., and Brown, D. D. (1980) Cell 19,

18,469-484

* J. Segall and R. G. Roeder, unpublished data.

RNA Polymerase 111 Transcription Factors 11991

13-25 22. Doering, J. (1977) Carnegie Inst. Wash. Year Book 76, 120-125 18. Bogenhagen, D. F., Sakonju, S., and Brown, D. D. (1980) Cell 19, 23. Kom, L. J., and Brown, D. D. (1978) Cell 15, 1145-1156

27-35 24. Clarkson, S. G., Kurer, V., and Smith, H. 0. (1978) Cell 14, 713- 19. Kressman, A., Hofstetter, H., DiCapua, E., Grosschedl, R., and 724

Birnsteil, M. L. (1979) Nucl. Acids Res. 7, 1749-1763 25. Bothwell, A. L. M., and Altman, S. (1975) J. Biol. Chem. 250, 20. DeFranco, D., Schmidt, O., and SOU, D. (1980) Proc. Nutl. Acad. 1451-1459

Sci. U. S. A . 77,3365-3383 26. Roeder, R. G. (1976) in RNA Polymerase (Losick, R., and Cham- 21. Matsui, T., Segall, J., Wed, P. A,, and Roeder, R. G. (1980) J. berlin, M., eds) pp. 285-329, Cold Spring Harbor Laboratory,

Biol. Chem. 255, 11992-11996 New York