Proc. Natl. Acad. Sci. USA Vol. 89, pp. 11292-112%, December 1992 Biochemistry

Yeast RNA polymerase II initiation factor e: Isolation and identification as the functional counterpart of human transcription factor IIB (Saccharomyces cerevisiae/in vitro transcription)

HERBERT TSCHOCHNER, MICHAEL H. SAYRE, PETER M. FLANAGAN, WILLIAM J. FEAVER, AND ROGER D. KORNBERG Department of Cell Biology, Fairchild Center, Stanford University School of Medicine, Stanford, CA 94305

Communicated by Michael J. Chamberlin, August 31, 1992 (received for review July 14, 1992)

Yeast RNA polymerase II initiation factor e ABSTRACT was purified to homogeneity and identified by biochemical criteria as the counterpart of human transcription factor RB. Factor e was essential for itition of transcription from yeast and mammalian promoters In a reconstituted yeast transcription system. Activity resided in a single polypeptide of -41 kDa, identified by peptide sequence analysis as the product of the SUA7 gene. Factor e interacted specifically with RNA polymerase II, consistent with a proposed role in determining the start site of transcription.

Promoter utilization by RNA polymerase II requires protein transcription factors (TFs), termed TFIIA, -B, -D, -E, -F, -G, -H, and -J (or BTF-1, -2, -3, etc.) from human cells (1-3); a, (3, y, 8,6E, and X from rat liver (4); and a, b, d, e, and g from Saccharomyces cerevisiae (Sa. cerevisiae) (5-9). Resolution of these factors and reconstitution of transcription with purified components are major objectives of current research, important for mechanistic and structural studies of transcription complexes. Yeast is advantageous for such studies because of its facility for biochemical and genetic analyses; results should be informative for mammalian systems since many aspects of RNA polymerase II transcription have been conserved across species (1, 4, 10). Activities that substitute for TFIIA and TFIID in the human system have been found in yeast extracts, leading to isolation and cloning ofthese factors (11, 12). Efforts to replace other human initiation factors with yeast proteins have been unsuccessful, raising the question of whether functional counterparts ofTFIIB, -E, -F, and so forth exist in yeast or whether the activities of these factors are distributed among yeast polypeptides in a different manner. The yeast SUA7 gene encodes a protein with 35% sequence identity to human TFIIB, and sua7 mutations alter the locations of transcription start sites at two promoters in vivo (13). Does the SUA7 gene product play the same functional role as TFIIB in transcription? We report here the purification of yeast RNA polymerase II initiation factor e and its identification as the SUA7 gene product and present biochemical evidence that factor e/SUA7 is the functional counterpart of human TFIIB. We further show that purified factore interacts specifically with RNA polymerase II, consistent with the idea, based on the effects of sua7 mutations and other work, that factor e/TFIIB plays a role in determining the start site of transcription through interaction with the polymerase (13).

EXPERIMENTAL PROCEDURES Reconstituted Transcription. Transcription reaction mixtures (25 Al) contained 20 mM Hepes-KOH (pH 7.6), 8 mM The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

MgSO4, 2 mM dithiothreitol (DTT), 12.5% (vol/vol) glycerol, 80-120 mM potassium acetate, 0.4 mM ATP, 0.4 mM CTP, 12 ,uM UTP, 7.5 tuCi of [a-32P]UTP (1 Ci = 37 GBq), 250 ng of template, purified yeast TFs, and RNA polymerase II. Templates were plasmids pGAL4CG-, pMLG- (14), p(GCN4)2ADHG- (15), or pPYKlG-, all of which contain the G- cassette of Sawadogo and Roeder (16). pPYK1Gcontains the Sa. cerevisiae PYKI promoter (from positions -221 to -16 relative to the ATG; ref. 17) fused to the Alu I site of the G- cassette (16), with the promoter-G- fusion inserted between the Pst I and BamHI sites of pBluescript KS (Stratagene). The initiation site is within nucleotides CAAG at positions -36 to -33 in the wild-type sequence (17); the G at -33 is replaced by C in pPYKlG-. Assays of highly purified factor e fractions (fraction VIVIII; Table 1) contained 25 ng of homogeneous yeast TFIID (18), along with 100 ng of yeast RNA polymerase II fraction IV, 20 ng of factor a fraction VI, and 1 Mg of factor b/g DEAE-5-PW fraction, prepared as described elsewhere (7, 8). Assays of crude factor e-containing fractions were supplemented with 1 Mg of the antiinhibitory factor f hydroxylapatite fraction (7). Reaction mixtures were incubated at 200C for 30 min and RNA was analyzed as described (14). Buffers. TP buffer contained 20 mM Tris acetate (pH 7.9), 20%o glycerol, 1 mM EDTA, 1 mM DTT, 0.01% Nonidet P-40, protease inhibitors, and the concentration of potassium acetate given in parentheses as mM. Buffer P contained 20 mM Tris acetate (pH 7.9), 10%6 glycerol, 7 mM magnesium acetate, 1 mM EDTA, 1 mM DTT, protease inhibitors (5), and the concentration of potassium acetate given in parentheses as mM. Purification of Factor e. The preparation of whole cell extract from yeast strain BJ926 and its fractionation on Bio-Rex 70, DEAE-Sephacel, and hydroxylapatite columns are described elsewhere (7, 15). Hydroxylapatite fractions containing the peak of factor e activity were pooled (Table 1, fraction IV) and dialyzed against TP(0) to the conductivity of TP(100). Insoluble material was removed by centrifugation in a Beckman 45 Ti rotor at 20,000 rpm at 40C for 20 min, and the supernatant was loaded at 0.5 ml/min onto a Bio-Gel SP-5-PW HPLC column (75 x 7.5 mm; Bio-Rad) equilibrated in TP(100). The column was washed with 10 ml of TP(100) and material was eluted with a 30-ml linear gradient from 100 to 600 mM potassium acetate in TP. Factor e activity was eluted at the conductivity of TP(330). Three SP-HPLC column runs were necessary to process 120 mg of fraction IV protein. Fractions containing activity were pooled (fraction V), diluted with TP(0) to the conductivity of TP(150), and applied at 0.4 ml/min to a TSK-heparin-5-PW HPLC column (75 x 7.5 mm; Supelco) equilibrated in TP(150). The column was washed with 10 ml of TP(100) and material was eluted with a Abbreviations: TF, transcription factor; DTT, dithiothreitol. 11292

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11293

Table 1. Purification of factor e from 2.5 kg of yeast Fraction Protein, Volume, Activity, Specific activity, Relative Recovery, No. Step mg ml units units/mg specific activity % I Whole cell extract 120,000 5400 II Bio-Rex 70 8,300 460 220,000 27 1 100 III DEAE-Sephacel 1,100 160 128,000 116 4 58 IV Hydroxylapatite 120 55 73,000 608 23 33 V Bio-Gel SP-5-PW 11 12 20,000 1,818 69 9 VI TSK-heparin-5-PW 6.1 5.6 12,800 2,102 79 6 VII Bio-Gel DEAE-5-PW 0.09 14.4 9,150 101,666 3800 4 VIII TSK-heparin-5-PW 0.03 1.6 3,300 106,451 3900 2 For factor e, 1 unit of activity is defined as the amount required to produce 0.1 fmol of accurately initiated t pts in the reconstituted transcription system. Factor e activity could not be detected in the whole cell extract due to the presence of inhibitors. Measurements of factor e activity in fractions II-VI are also affected by the presence of inhibitors (see text).

linear gradient (36 ml) to TP(850). Factor e activity was eluted at 450 mM potassium acetate. Peak fractions were pooled (fraction VI), dialyzed against TP(0) to the conductivity of TP(50), and loaded at 0.4 ml/min onto a Bio-Gel DEAE-5-PW HPLC column (75 x 7.5 mm; Bio-Rad) equilibrated in TP(50). The column was washed with TP(50) and the factor e-containing fractions of the flow-through were pooled (fraction VII) and subjected to chromatography on the TSK-heparin5-PW column as above to yield fraction VIII. Reverse-phase HPLC was carried out on a Hi-Pore RP-304 C4 column (4.6 x 250 mm; Bio-Rad). About 4 ,ug of fraction VIII was adjusted to 2% (vol/vol) trifluoroacetic acid, loaded onto the column at 1 ml/min, and eluted at 1 ml/min with a linear gradient (60 ml) from 0o to 80% (vol/vol) acetonitrile in 0.1% trifluoroacetic acid. Factor e was eluted in a single fraction (1 ml) at 61% acetonitrile. Reverse-phase-purified factor e (215 pmol) was digested with trypsin and three derived peptides were isolated by reverse-phase HPLC and subjected to N-terminal sequencing. Gel Electrophoresis of Protein-DNA Complexes. Gelelectrophoretic-mobility-shift assays were performed as described (19) with an adenoviral major late promotercontaining DNA fragment (6), and TFs as indicated. ProteinDNA complexes were fractionated by electrophoresis in 5% polyacrylamide gels in 0.5x TBE buffer (31). Yeast TFIIA was prepared from whole cell extract by chromatography on DE52 (Whatman; ref. 5), Bio-Rex 70 (Bio-Rad), and heparinand Q-Sepharose (Pharmacia) (Y. Li, N. L. Henry, and M.H.S., unpublished data). The Q-Sepharose fraction contained the large subunit of yeast TFIIA (TOAl), as determined by Western blot analysis with anti-TOAl antiserum, and could be replaced by purified bacterially expressed yeast TFIIA (11) in the gel-mobility-shift assay (Y. Li and W.J.F., unpublished data). Human TFIIB was purified from a bacterial lysate (20) by chromatography on DE52, phosphocellulose P11 (Whatman), and hydroxylapatite and was determined to be >95% pure by densitometry of Coomassie blue-stained SDS/polyacrylamide gels (W.J.F., unpublished data). RNA Polymerase U Affinity Column. Purified RNA polymerase 11 (0.09 mg) was mixed gently for 2 hr at 40C with monoclonal antibody 8WG16-Sepharose beads (0.25 ml; 21) equilibrated in 20 mM Tris acetate, pH 7.9/0.4 M potassium acetate/7% glycerol/i mM EDTA/5 mM DTT. The beads were then washed batchwise three times with 10 ml of 20 mM Tris acetate, pH 7.9/1 M potassium acetate/7% glycerol/i mM EDTA/5 mM DTT and then three times with 10 ml of buffer P(100). A sample of factor e fraction V (Table 1) was dialyzed to the conductivity of TP(50) and applied to a DEAE-5-PW column as above, and 20;Lg of the flow-through fiaction (0.5 ml; containing -4 MLg, or 0.1 nmol, factor e) was added to the polymerase beads in P(100) (total volume, 2 ml) and mixed gently at 40C for 1 hr. Bovine serum albumin (0.1

mg; Sigma, protease-free) was also added to stabilize factor e activity. The slurry was placed in a Poly-Prep column (Bio-Rad) and washed with 1.25 ml of P(100) (flow-through fraction). Material was eluted with 1.25-ml volumes of P(200), P(400), and P(750). After washing the column with P(1000), RNA polymerase H was then eluted with P(100) containing 30% glycerol. About 80% of the loaded polymerase was recovered. A control column was prepared and loaded with factor e, and material was eluted exactly as above, but RNA polymerase II was omitted.

RESULTS Isoltion of Factor e aid Its e fcat s the SUA7 Gene Product. Purification of factor e from whole cell extract was guided by a functional assay, based on its requirement for CYC1 promoter-directed initiation by RNA polymerase II in a reconstituted transcription system consisting of homogeneous yeast TFIID (18), RNA polymerase II, factor a, and a fraction enriched for factors b and g (7, 8). Inclusion of factor f was necessary for detection of factor e activity at early stages of purification but not when nearly homogeneous factor e was used (7). Factor e was purified in seven chromatographic steps (Table 1 and Fig. 1). Most enrichment was achieved through binding of the factor to DEAE-cellulose at an early stage in the purification, possibly in association with RNA polymerase II and factors b and g (7), and lack of binding to DEAE at a later stage. Lack of binding to DEAE is a distinctive property shared with mammalian TFIIB (20). The yield of factor e activity in transcription could only be roughly estimated because activity was undetectable in the starting extract and because inhibitory contaminants persisted until late in the purification. Factor e activity in crude fractions was stabilized by the presence of 0.3 M potassium acetate

45-

22-

FIG. 1. Purification of factor e. Protein fractions (Table 1) were analyzed in an SDS/12% polyacrylamide gel (Coomassie bluestained gel shown). Lanes: 1, protein standards (from Bio-Rad; masses in kDa indicated at left); 2-8, fractions II (60 pg), III (21 pg), IV (21 pg), V (7.2 pmg), VI (6 jug), VII (0.6 Ag), and VIII (0.6 jIg), respectively. Band due to the 41-kDa factor e polypeptide is indicated by arrow.

Proc. Natl. Acad. Sci. USA 89 (1992)

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11294

(P.M.F., unpublished data). There were at least two inhibitory components in fraction VI, only one of which was counteracted by factor f (H.T., unpublished data). Activity cochromatographed with a 41-kDa polypeptide in the final step of purification (Fig. 2). Further evidence that factor e activity resided in the 41-kDa polypeptide was obtained by reverse-phase HPLC (Fig. 3). After renaturation, activity was found in a sharp peak coincident with the 41-kDa polypeptide (fraction 25). The 41-kDa polypeptide cosedimented with the activity in a glycerol gradient at a rate indicative of a native mass of 40-90 kDa (H.T., unpublished data). The generality of a requirement for factor e in transcription was tested with the Sa. cerevisiae PYKJ promoter, Schizosaccharomyces pombe (Sc. pombe) ADHI promoter, and adenoviral major late promoter: in each case, accurate initiation required the purified factor (Fig. 4). The same result was obtained with the Sa. cerevisiae GALIO and ADCO promoters (7). The sequences of three tryptic peptides of factor e, NHRGPNLNIVLTXPEXK-COOH (where X indicates an unidentified residue), NH-VGQTLQVTEGTI-COOH, and NHILYEH-COOH, were identical to deduced amino acid sequences in the SUA7 gene at residues 14-28, 298-309, and 315-319, respectively (13). Moreover, SUA7 protein purified

A 80o >5

.B: C

60-

.2c 40-

(U20vIr

21 22 23 24 25 26 27 28 29 30 Fraction

B M 20 21 22 23 24 25 26 27 28 29 30 31 32 33

A 31 32 33 34 35 36

97-

-

45 -

W

31 -

_

22-

w

*@lb

FIG. 3. Association of factor e activity with 41-kDa polypeptide in the denatured state. Samples (0.25 ml) of reverse-phase HPLC column fractions indicated were evaporated to dryness and either dissolved in 50 Al of 6 M guanidine hydrochloride, dialyzed overnight against 200 ml of TP(100) buffer, and assayed (5 ,ul) for transcription (radioactivity in full-length transcripts plotted as percentage of activity recovered from all fractions) (A) or dissolved in SDS gel loading buffer and subjected to electrophoresis in an SDS/12% polyacrylamide gel (silver-stained gel shown) (B). Molecular masses in kDa are shown.

B onnnn

15000-

ECL 10000-

*5 5000-

28 29 30 31

32 33 34 35 36 Fraction

30 31 32 33 34 35 36

C

4

97 -

45 _

31

_

i

from an overproducing bacterial strain exhibited factor e activity in reconstituted transcription assays (E. Maldonado and H.T., unpublished data). We conclude that SUA7 encodes factor e. Interaction of Factor e with RNA Polymerase II. Factor e (derived from fraction V) was incubated with Sepharose beads containing RNA polymerase II bound to immobilized antibodies directed against the C-terminal repeat domain of the polymerase (21). Nearly all factor e transcription activity bound to the beads (Fig. SA; compare load with flow-through from the polymerase column), whereas the activity flowed through a control column of immobilized antibodies lacking polymerase. Elution with 0.4 M and 0.75 M potassium acetate yielded 28% of the factor e activity applied to the polymerase 2

1 -e +elr

-

3

4

+-e+elF-e-+e

:s

1~~~~~~ _

q

4::

4 A

mv1R1S

FIG. 2. Factor e activity copurites with a 41-kDa polypeptide in the native state. Fractions from the final TSK-heparin-5-PW column (Table 1) were analyzed for transcription activity (4 Al of fractions indicated; autoradiogram of transcripts in A and radioactivity in transcripts plotted in B) and by electrophoresis in an SDS/12% polyacrylamide gel stained with silver (50 ,ul of fractions indicated in C). Molecular masses in kDa are indicated.

*

*

FIG. 4. Generality of factor e requirement in transcription. Reconstituted transcription with purified yeast factors with (+) or without (-) factor e (5 ng of frac~ tion VIII) as indicated, on templates with the Sa. cerevisiae CYCI (lanes 1) and PYKI (lanes 2), adenoviral major late (lanes 3), or Sc. pombe ADH (lanes 4) promoters. Autoradiogram reveals specifically initiated transcripts.

Proc.

A

RNA pol 11 column boad

-e

ft.

f.

P 400

4f1 e12 47

.a

P 750

1[2

4

polymerase column (along with some polymerase; lanes 3 and 4) but not from the control column (lanes 9 and 10). Polymerase bound factor e selectively, as shown by the failure of most contaminants in the factor e fraction (which were, judging from their chromatographic behavior, basic like factor e) to bind to the polymerase column (compare lanes 2 and 7; additional bands in lane 7 not visible in lane 2 were contributed by added bovine serum albumin). The affinity of factor e for polymerase was evidently substantial, as the concentration of factor e polypeptide in the binding reaction

was

_1

E 0

was, at most, -50 nM, similar to the concentrations used in transcription reactions. Since the factor e preparation applied

0

to the polymerase column contained additional yeastproteins, it is possible that one or more ofthem assisted factor e in binding to polymerase.

B

Interaction of Factor e with TFIIA and TFlD on Promoter DNA. Electrophoretic-mobility-shift analyses have revealed specific binding of human TFIIB with TFIIA-TFIIDpromoter DNA complexes (19, 22, 23). Analogous experiments with factor e gave similar results (Fig. 6). A DNAprotein complex whose formation required yeast TFIIA, TFIID, and factor e (lanes 8 and 9) had an electrophoretic mobility indistinguishable from that of a TFIIA-TFIIDhuman TFIIB-DNA complex (lanes 10 and 11).

6645-

DISCUSSION Because of its instability, low abundance, and association with inhibitors of transcription, isolation of factor e in pure form had presented one of the main obstacles to reconstitution of promoter-directed yeast RNA polymerase II transcription with purified components. This obstacle has finally been overcome (this work and refs. 7-9), especially since relatively large amounts of factor e can now be obtained by expression in bacteria and purification by a simplified version of the present procedure (H.T. and W.J.F., unpublished

I - factor e ..Www:. qh. %

am

31

11295

Analysis by SDS/PAGE gave corresponding results (Fig. 5B). The 41-kDa polypeptide was nearly absent from the flow-through of the polymerase column (lane 7) but was prominent in the flow-through of the control column (lane 8). The polypeptide was eluted with potassium acetate from the

control column

P 400 P 750

Nati. Acad. Sci. USA 89 (1992)

-

.--1

I

__

i

2 2 -*Fe __-

1

3

2

5

4

6

7

8

9

10

data).

FIG. 5. Interaction of factor e with RNA polymerase II. Factor e was loaded on a column ofRNA polymerase or on a control column

The biochemical properties shared by factor e and human TFIIB proteins can be summarized as follows. Factor e and TFIIB are essential for promoter-dependent transcription initiation in the yeast and human (HeLa) systems, respectively. The factor e polypeptide is similar in size to TFIIB and exhibits chromatographic behavior similar to that of TFIIB. Factor e causes a shift in the electrophoretic mobility of a TFIIA-TFIID-promoter DNA complex that is indistinguishable from that caused by TFIIB. These similarities, combined with the sequence homology between factor e (SUA7) and

lacking polymerase. The material loaded, flow-through (f.t.), and material eluted with 400 and 750 mM potassium acetate (P400 and P750, respectively) were analyzed in transcription assays (2 or 4 ;LI of the fraction indicated) (A) and in an SDS/12% polyacrylamide gel, stained with silver (0.25 ml of each fraction) (B). Molecular masses in kDa are shown in B.

column (Fig. SA), but no activity was eluted from the control column. hIB

-

-

10 ng

5

.5

e

ylIA

-

u1

-

+

-

yllD

+

+

-+

+

+

+

+

*1

,A ""-"

.

-W.

-* -a

-

1

1 .2

3

2

3

5 4. 64

5

6

7

8

9 9

0 10

11

yllD

+

yllA

yllD

+

yllA

free probe

+

e/hIlB

FIG. 6. Interaction of factor e with a TFIID-TFIIApromoter DNA complex. Yeast TFIIA (yIIA, 2 Al), TFIID (yIID, 5 ng; ref. 18), factore fraction VIII (e), and human TFIIB (hIIB) were added as indicated. Bands formed in a nondenaturing polyacrylamide gel by TFIIA-TFIID-DNA (yIID + yIIA) and by TFIIA-TFIIDfactor e- or human TFIIB-DNA complexes (yIID + yIIA + e/hIIB) were visualized by autoradiography.

112%

Biochemistry: Tschochner et al.

TFUIB genes, identify factor e as the physical and functional counterpart of human TFIII3 in yeast. Results from electrophoretic-mobility-shift (19, 22, 23) and template-challenge (24) experiments have indicated that TFIIB/rat a enters an initiation complex after TFIID but before RNA polymerase II, prompting speculation that TEIB might "bridge" between TFIID and the polymerase and thus establish the distance between the TATA element and start site of transcription (22). Our finding that factor e associates with polymerase under conditions ofionic strength and magnesium concentration used for transcription is compatible with that idea. Effects of sua7 mutations provide supportive evidence as well (13), but mutant SUA7 proteins have not yet been tested for their ability to bind to polymerase in vitro. Mutations in other yeast genes also perturb start-site selection (25-30); the question, therefore, remains whether factor e/SUA7 protein is primarily responsible for setting the distance between TATA box and initiation site through direct interaction with RNA polymerase II or whether it is just one of many factors that influence the process. The question might be answered by exchange of factor e and TFIIB between yeast and mammalian systems in vitro, as the distance from TATA element to transcription start site differs markedly between (those systems, but such exchange of factor e and rat liver factor a (rat TFIIB) failed to yield detectable transcription (J. W. Conaway and H.T., unpublished data). An alternative swap between the Sa. cerevisiae and newly devised Sc. pombe system (in which initiation occurs at the same distance from the TATA element as in mammalian cells; ref. 15) awaits isolation of an Sc. pombe TFIIB counterpart. Finally, factor e variants encoded by sua7 alleles can now be purified from yeast or bacteria and tested in our reconstituted transcription system to delineate functional domains involved in transcription and polymerasebinding activity and to investigate whether other factors contribute to the effects of SUA7 mutations on start site selection (13). We thank N. Thompson for 8WG16 antibody, S. Hahn for purified recombinant yeast TFIIA and antibodies against TFIIA, and D. Reinberg for the human TFIIB clone. H.T. was supported by a fellowship from the Deutsche Forschungsgemeinschaft, M.H.S. was supported by a fellowship from the American Cancer Society, and W.J.F. was supported by a Medical Research Council of Canada studentship. Costs ofthis research were paid from National Institutes of Health Grant GM36659 (R.D.K.).

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