A complex that contains proteins binding to the PSE and TATA sites in a human U6 small nuclear RNA promoter

Gene, 148(1994)269-215 0 1994 Elsevier Science B.V. All rights reserved.

269

0378-l 119/94/$07.00

GENE 08212

A complex that contains proteins binding to the PSE and TATA sites in a human U6 small nuclear RNA promoter (Eukaryotic promoters; RNA polymerase III; snRNA gene transcription factors)

Randal S. Goomer*,

Olgui Urso and Gary R. Kunkel

Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA Received by J.A. Gorman:

22 November

1993; Revised/Accepted:

23 May/24

May 1994; Received at publishers:

27 June 1994

SUMMARY

The proximal promoter of a human U6 small nuclear RNA (snRNA)-encoding gene contains two separate elements, the proximal sequence element (PSE) and the TATA box. We investigated the interaction of the PSE- and TATA-binding proteins (PBP and TBP) with normal and mutant U6 proximal promoters using an electrophoretic mobility shift assay. We detected a complex containing both PBP and TBP bound to the wild-type U6 promoter. Efficient formation of the triple complex was dependent on the presence of the PSE and the TATA box on the template DNA. Mutant U6 promoters containing an increased spacing between the PSE and TATA box of 5 or 10 bp were impaired in the ability to form a complex that includes TBP. We infer from these results that PBP and TBP interact when their binding sites are properly positioned in a U6 gene promoter.

INTRODUCTION

Promoter elements in the 5’ flanking sequences of vertebrate U6 snRNA genes are very similar to those of RNA polymerase-II-transcribed genes. The proximal promoter of U6 consists of a TATA-box sequence Correspondence to: Dr. G.R. Kunkel,

Department

Biophysics,

College

Texas A&M

University,

USA. Tel. (l-409) 845-6257; Fax (l-409) e-mail: [email protected] *Present

address:

Department

of Biochemistry

Station,

and

TX 77843-2128,

845-9274;

of Neurobiology,

The Scripps

Research

Institute, 10666 N. Torrey Pines Road, La Jolla, CA 92037, USA. Tel. (1-619) 554-3634; Fax (1-619) 554-6660. Abbreviations: Ab, antibody(ies); bp, base pair(s); BSA, bovine serum albumin; de, decreased; dpm, disintegrations per min; DTT, dithiothreitol; EMSA, electrophoretic mobility-shift assay; h, human; HEPES, 4-[2-hydroxyethyll-1-piperazine-ethanesulfonic acid; in, increased; PA, polyacrylamide; PAGE, PA-gel electrophoresis; PBP, PSE-binding protein; PCR, polymerase chain reaction; P-D complex, PBP-DNA complex; P-D-T complex, PBP-DNA-TBP complex; pol, RNA polymerase; PSE, proximal sequence element; re-, recombinant; SDS, sodium dodecyl sulfate; snRNA, small nuclear RNA; TBP, TATA-binding protein; wt, wild type; y, yeast. SSDI 0378-l

119(94)00423-4

(-29TATATA-24 m . a human gene) and a PSE sequence (-66CTTACCGTAACTTGAAAGTA-47 in a human gene). The U6 TATA element is required for transcription by RNA polymerase III (pol III) and its elimination results in transcription exclusively by pol II (Mattaj et al., 1988; Lobo and Hernandez, 1989). It has been shown recently that TATA-binding re-protein (TBP) is required for U6 transcription in vitro (Margottin et al., 1991; Simmen et al., 1991; Lobo et al., 1991). Furthermore, a role for TBP in the transcription of 5s and tRNA genes by pol III and rRNA genes by pol I has been shown (White et al., 1992; Cormack and Struhl, 1992; Schultz et al., 1992; Taggart et al., 1992; Lobo et al., 1992; White and Jackson, 1992b; Kassavetis et al., 1992; Comai et al., 1992). TBP has emerged as a general transcription factor for transcription by all three RNA polymerases (Sharp, 1992; White and Jackson, 1992a; Rigby, 1993; Hernandez, 1993). The vertebrate pol-II-transcribed genes that encode the abundant spliceosomal snRNAs (UI, U2, U4 and U5), do not require the presence of a TATA box. Instead the proximal promoters of these genes contain the PSE that

270 is necessary

for

transcription

(Skuzeski

Ciliberto

et al., 1985; Mattaj

Murphy

et al., 1987). In addition,

efficient

transcription

Hernandez, groups

have identified

region and is required (PBP, PTF or SNAP,; et al., 1992; Murphy Sadowski

a PSE is required I/6 gene (Lobo

and Pederson,

a protein Waldschmidt

reduction

in transfected Goomer

of a rigid others

cells (Mattaj

spacing

et al., 1993;

et al., 1993).

changes

in transcriptional

and Kunkel,

in the PSE

et al.. 1991; Simmen

the TATA results

in a

efficiency in vitro and

have hypothesized

the two elements, that

the protein

we and

factors

that

bind the PSE and the TATA box may interact with one another, either directly or indirectly. Using a gel mobilityshift analysis, we detected a complex containing both the PBP and recombinant human TBP on the U6 promoter. DNA fragments containing spacing changes that greatly reduce transcriptional efficiency in vitro supported a reduced level of this complex. Thus we conclude that these transcription factors interact when bound to the U6 gene promoter.

RESULTS

u-TBP

Ab was added

(Fig. 2, lanes 2 4). Addition control

(a-TFIIB)

detected

to the binding of another

rabbit

complex

upon addition

Ab, even when these experiments

out with purified

Ah as ;I

had little effect (Fig. 2, lanes 5- 7). We

no discrete supershifted

of a-TBP

reaction

Ab (results

not shown).

were carried Although

this

experiment did not rule out the possibility of a distinct PBP polypeptide that shares a TBP epitope, it is likely that

the P-D complex

contains

TBP.

Furthermore.

in PSE-binding

not bind the TATA box, since no difference plex formation probes

was detected

were compared

when

protein

we does

in P-D com-

wt and

TATAmut

(Fig. 5. lanes 2 and 6).

et al., 1988; Lobo et al., 1991;

1992). Based upon the requirement between

rabbit

infer that this TBP present

between

box and the PSE in the U6 gene promoter drastic

and

U6 gene transcription

et al., 1992; Wanandi

of spacing

for

1988). Several

that binds

for efficient

et al.. 1993; Bernues

Introduction

1984:

et al.. 1985; Ares et al., 1985:

of a human

1989; Kunkel

et al.,

AND DISCUSSION

(a) Detection of a complex on the human U6 proximal promoter that contains PBP Fig. 1A depicts the labeled probes used in these experiments. Probe A contained the wt U6 sequence from -84 to + 111 that includes the U6 proximal promoter along with most of the U6 coding region. Probe B contained exclusively 5’ flanking sequences from - 148 to - 1. Where tested, each probe gave essentially identical results in the experiments reported here. A partially purified fraction of PBP from human 293 cells retarded the mobility of the wt probe A in a nondenaturing PA gel (the P-D complex; Fig. lB, lane 2). The shifted complex could be competed effectively by an unlabeled U6 plasmid containing the PSE (the wt or the TATAmut plasmids; Fig. 1A) but not a plasmid that contained a mutated PSE sequence (PSEmut) (Fig. 1B; compare lanes 3 and 4 with lane 5). It has been reported recently that a different preparation of HeLa cell PSE-binding protein, SNAP,, contained TBP (Sadowski et al., 1993). We explored whether the P-D complex could be disrupted by addition of an Ab that reacts against human TBP. Indeed, the ability to detect the P-D complex was drastically reduced when

(h) Formation of a complex containing PBP and re-TBP on the human U6 proximal promoter Inclusion

of recombinant

human

TATA-binding

pro-

tein (re-hTBP) along with PBP resulted in the formation of another complex of reduced mobility on the wt probe (the P-D-T complex; Fig. lB, lane 6). The P-D-T complex, along with the P-D complex could be competed away by the wt or the TATAmut U6 plasmids but not the PSEmut plasmid (Fig. 1B; compare lanes 7 and 8 with lane 9). The ability of PSE-containing plasmids and not the PSEmut plasmid to efficiently compete both the P-D and P-D-T complexes provides evidence that the PSE-binding activity is present in both complexes. It is curious that the PSEmut plasmid DNA did not compete the P-D-T complex whose presence depended on the addition of TBP. since this plasmid template contains an intact TATA box. Large amounts of recombinant TBP were used in these binding assays, and we calculate that the molar concentrations of TBP and Uh promoter were nearly equivalent when competitor plasmid DNA was included in the reaction. The band marked with an asterisk represents binding of nonspecific protein(s) present in the PBP preparation, since it was not competed by relatively high levels of the unlabeled specific competitor (Fig. 1B). The collection of heterogeneous bands marked with an open bracket was observed only when re-TBP was present in the reaction mixture and was not efficiently reduced by specific competitors (Fig. lB, lanes 7-9). These broad bands were observed only when TATA-containing templates were used in the presence of TBP, and their intensities were enhanced when PBP was included in the reaction also. One implication of this observation is that these bands represent TBP-DNA complexes that are facilitated to form in the presence of PBP. Yeast TATA-binding protein (re-yTBP) also formed a distinct, slower-mobility complex when incubated with the human PBP preparation and the U6 DNA probe. The PBP-DNA-re-yTBP complex had a slightly greater mobility than the PBP-DNA-re-hTBP complex (Fig. 3:

A,1S wtU6

-84

-148

probe A probe 8

l

1 PSE

TATA

P-D-T L P-D -

=

TATAmut

PSEmut *inP series

D-

spacing mutants PBP (P)

inP3 PSE

TATA

re_hmpm

-

++++++++

-

-

-

-

-

++++

123456789

inP5 s?“-=

TATA

inP1 Cl

Probe: r

A

TATA

Fig. 1. Detection of protein-DNA complexes on the human U6 promoter. (A) U6 promoter constructs used in EMSA. The TATAmut and PSEmut promoters contained clustered point mutations to disrupt the TATA box and the PSE, respectively (Kunkel and Danzeiser, 1992). The inP3, inP5 and inPl0 mutant constructs contained increased spacing between the PSE and the TATA box of 3, 5 or 10 bp, respectively (Goomer and Kunkel, 1992). (B) EMSA: The PBP fraction (2 pg protein) was incubated with a radiolabeled wt U6 probe A for 30 min and electrophoresed on a 4% PA non-denaturing gel to separate complexes. In lanes 2 to 5, PBP (2 ug) was incubated with the radiolabeled wt U6 probe in the presence of 1 ug of the specific and non-specific unlabeled competitor DNAs (approx. lO%fold molar excess). In lanes 6 to 9, PBP (2 ug) and re-hTBP (0.08 ug) were incubated with radiolabeled wt U6 probe in the presence of 1 ug (approx. lOO-fold molar excess) of the same competitor DNAs. The asterisk marks a non-specific complex. The open bracket identifies a series of bands present in reactions containing re-hTBP. Methods: The radiolabeied U6 promoter probes used in this study were generated by PCR. For probe A, the ‘U6’ primer (5’-TATGGAACGCTTCAC) was end-labeled and paired with the ‘Rsa’ primer (S-ACAAAATACGTGACGTAGAA). This probe was truncated by digestion at position - 84 with Draf. For probe B, the ‘Rsa’ primer was end-labeled and paired with the ‘cU6-1’ primer (T-GGTGTTTCGTCCTTTCCACA). Probes were purified by gel electrophoresis and dissolved in 10 mM Tris/l mM EDTA (pH 7.5) to give 20000 dpm/ul (approx. spec. act. 5 x lo6 dpm/pmol). PBP was purified from 20 litres 293 cells (a human embryonic kidney cell line) grown in suspension in Joklik’s minimum essential medium containing 5% calf serum and 1% newborn calf serum. SlOO extract (approx. 14-17 mg/ml; Goomer and Kunkel, 1992) was loaded onto a phosphocellulose (Whatman Pll) column (10 mg protein per ml of resin) equilibrated with buffer D (20 mM HEPES pH 7.9/20% glycerol/O.2 mM EDTA/O.S mM DTT/O.I M KCl). The transcriptionally active fractions (present in the 0.1 to 0.6 M KC1 eluate at approx. 2.5 mg/ml protein concentration) were dialyzed against buffer B (50 mM TrisHCl pH 7.9/0.1 mM EDTA,/2 mM DTT) containing 50 mM (NH&SO, and loaded onto a DEAE-Sephadex column equilibrated with the same buffer ( 1mg protein/ml of resin). PBP activity eluted with buffer B containing 0.15 M (NH4)$04 and was dialyzed against buffer D. P&E-binding activity was monitored by EMSA as described below. This sample (approx. 10 mg protein) was loaded onto a heparin-agarose column ( 10 ml resin) equilibrated with buffer D. Fractions containing the PSE-binding activity were step-eluted between 0.2 M and 0.35 M KC1 and dialyzed against buffer D. The heparin eluate (approx. 0.5 mg/mI protein) was loaded onto a 100 ml Sephacryl-HR2~ (Pha~acia) column equilibrated with buffer D. All of the FSE-binding activity was present in fractions that eluted immediately after the void volume (approx. 0.2 mg/ml protein). Fractions were pooled and quick frozen in liquid nitrogen and stored at -80°C. Purified re-hTBP was the gift of the D.D. Peterson laboratory (Department of Biochemistry and Biophysics, Texas A&M University). The cDNA for hTBP (Peterson et al., 1990) was subcloned into the pET3c vector. The purity of the hTBP preparation was estimated to be approx. 50-60%, as determined by SDS-PAGE. The protein was stored in TMO.1 buffer (50 mM TrisHCl pH 7.9/i mM EDTA/12.5 mM MgCI,/l mM DTT/ZO% glycerol/100 mM KCl) at -80°C in individual-use aliquots. Binding reactions for gel mobility shift assays contained about 4 fmol of radiolabeled probe A (20000 dpm)/lO ul of Buffer A (25 mM HEPES pH 7.9/5 mM MgC1,/0.5 mM EDTA/SO mM KCl/0.5 mM DTTjtOX glycerol/O.5 ug BSA)/SOOng poly [d(G-C)]/l ug of nonspecific or specific competitor plasmid DNA and the indicated proteins in a 25 ul total reaction volume. The reactions were incubated for 30 min at 30°C and electrophoresed on a 4% polyacrylamide gel for 2.5 h at 200 V (constant voltage) in TGEM (25 mM TrisCl pH 8.3/0.19 M glycine/l mM EDTA/2 mM MgCl,) with 10% glycerol and 0.1% NP40 (Nonidet P-40). Gel electrophoresis was performed in 0.5 x TGEM buffer. Dried gels were autoradiographed with an intensifying screen.

compare lanes 6-8 and 10-12). This difference in the mobility of the P-D-T complex due to the addition of re-yTBP versus re-hTBP is consistent with the smaller mass of yTBP (27 kDa) versus hTBP (38 kDa) (Hahn et al., 1989; Peterson et al., 1990; Kao et al., 1990). We tested the dependence of the P-D-T complex on the presence of the TATA box in the human U6 promoter.

A much reduced ratio of the amount of complex with the mobility of P-D-T relative to P-D was formed on a radiolabeled TATAmut probe (Fig. 4, lanes 7-10; also see Fig. 5, lane II). When a very high amount of re-hTBP was present in the binding reaction (approx. 0.34 ug; Fig. 4, lane lo), we observed a band of reduced mobility. This slowest mobility complex could be a P-D-T com-

272

antibody:

_ P-D-T

top -

P-D !z’

c

P-D-

re-yTJ3P

*lz

-

-

-

-

+

+

+

+

-

re_n-t-np -

-

-

-

-

_

_

_

+

+

+

+

9

10

11

12

12345678 Fig. 3. EMSA of the slow mobility human

or yeast TBP is bound

are described was incubated

D-

of polyclonal

the P-D complex. bated with approx.

Ab against

hTBP

A radiolabeled wt Uh probe 2 ug PBP protein preparation

inhibits

formation

of

B (Fig. IA) was incuwith or without rabbit

Ab as designated above each lane. The sample loaded in lane I contained no Ab. Samples loaded in lanes 224 contained 0.1 ~1. 0.3 1.11and 1 ~1, respectively. of rabbit Ab that binds to hTBP. Samples loaded in lanes 557 contained 0.1 pl, 0.3 ul and 1 ul of rabbit Ab that binds to human

TFIIB,

for EMSA

used as a control

were as described

for this experiment.

in the legend

Conditions

to Fig. I. except

the position

used

that the

samples contained 1 ug poly [d(G-C)]:2 mM MgClz:5 mM Bands labeled with the asterisk identify non-specific complexes. denotes

-

( P-D-T) complex is different when

to the promoter.

Conditions

in the legend to Fig. 1. A radiolabeled with an increasing amount of added

for EMSA

wt C’h probe A PBP: Lanes: 1, 5

and 9, no PBP; 2, 6 and 10, approx.

0.5 ug PBP; 3. 7 and

1.25 pg PBP: 4, 8 and

2 pg PBP.

12: approx.

-

I I: approx.

The concentration

of

re-yTBP was kept constant in lanes 558 at approx. 0.04 pg. In lanes 9 to 12 a constant amount of re-hTBP was added to each of the EMSA

1234567 Fig. 2. Addition

-

DTT. ‘Top’

of the well.

plex, implying that the PBP bound on the TATAmut promoter can tether TBP into a P-D-T complex. On the wt promoter we observed that the predominant shifted complex corresponded to P-D-T after a large amount of TBP was titrated into the binding reaction (Fig. 4, lanes l-6). Interestingly, the total amount of P-D plus P-D-T complexes was increased in the presence of higher concentrations of TBP. Possibly, TBP facilitates the binding of PBP to the promoter. Several independent indirect observations identify the P-D-T complex as containing both the TBP and PBP bound to the U6 proximal promoter. (i) The observed expected difference in the mobility of the re-yTBP and re-hTBP dependent slow mobility complexes provides evidence that TBP is present in the P-D-T complex (Fig. 3). (ii) The slow mobility complex was dependent on the TATA box sequence. The P-D-T complex was detected readily on the normal promoter, while, at the same concentration of TBP, a very low level of P-D-T

(approx. 0.08 ug re-hTBP). The D-yT band represents a complex formed solely due to the incubation of re-yTBP with DNA. A smear present in lanes containing

re-hTBP

(lanes 9912) is marked

by an open bracket.

Purified re-yTBP was provided by the D.O. Peterson laboratory (Department of Biochemistry and Biophysics, Texas A&M University) and was estimated SDS-PAGE.

complex

to bc approx.

was observed

80 90%

pure,

on the TATAmut

as determined

probe

by

(Figs. 4

and 5). (iii) Increasing the concentration of TBP in the binding reaction resulted in an increased ratio of the P-D-T to P-D complexes, implying a precursor-product relationship between PBP-DNA and PBP-DNA-TBP complexes (Fig. 4). Taken together, the above observations imply that the complex identified as P-D-T contains both PBP and TBP bound to the U6 proximal promoter. (c) Spacing mutants that result in a greatly reduced level of transcription in vitro are also deficient in the ability to support formation of a complex containing both PBP and TBP In a previous study we had either increased (inP constructs) or decreased (deP constructs) the spacing between the PSE and TATA box by 1, 2, 3, 5 or 10 bp. Mutant promoters containing an increased spacing (of 5 or more bp) between the PSE and the TATA box supported a greatly reduced level of U6 specific transcription in vitro (Goomer and Kunkel, 1992). In order to investigate the stable binding of transcription factors on these mutant U6 proximal promoters, we prepared radiolabeled DNA fragments from these spacing change constructs (Fig. 1A). The specific radioactivity of each probe

213

Probes: P-D-T L P-D -

P-D-T L P-D -

c

c

**-

DPBP (P) l-BP(T)

-

+++++

++++

1

-++++ 23456

++++ 78910

I

I

I

TATAmut

wt promoter

Probe:

Fig. 4. Efficient detection dependent TATAmut

D-

of the slowest

mobility

complex

(P-D-T)

is

on the presence of a TATA box in the U6 probe. The wt or probes were incubated with a constant amount of PBP and

an increasing concentration of re-hTBP, EMSA was as described in the legend to Fig. 1. The experiments using the wt probe (lanes l-6) and TATAmut

probe

(lanes

7710) were carried

out at different

times. In

lanes 2 to 10 a constant amount of PBP (2 ug protein) was incubated with the probe. Lanes 3 and 7 contained 40 ng re-hTBP, lanes 4 and 8 contained 80 ng re-hTBP; lanes 5 and 9 contained 0.17 ug re-hTBP; and lanes 6 and 10 contained 0.34 ug re-hTBP. The band marked by the asterisk only

indentifies

in the wt probe

denoted

a non-specific reactions

complex.

containing

A diffuse band re-hTBP

(lanes

present 3-6)

is

by an open bracket.

was identical, since the same kinased oligodeoxynucleotide was used for each preparation. Fig. 5 shows the binding of PBP or PBP and TBP to these probes. A stable P-D complex was formed on all constructs tested, i.e., the wt, inP3, inP5, inPl0 or TATAmut DNA fragments (Fig. 5, lanes 2-6). However, the formation of the P-D-T complex was affected by a change in spacing between the two elements and was dependent on the presence of the TATA box. Relatively equal ratios of P-D to P-D-T complexes were detected on the wt and inP3 probes (Fig. 5, lanes 7, 8). However, for the inP5 and inPl0 probes, the ratio of the P-D to P-D-T complexes was much greater (Fig. 5, lanes 9, 10). The P-D-T complex was barely detectable on the TATAmut promoter probe in this experiment (Fig. 5, lane 11). In addition, we examined the formation of proteinDNA complexes on probes containing a decreased spacing between the PSE and the TATA box (deP5 or dePl0 constructs from Goomer and Kunkel, 1992), and found no change in the amount of P-D-T or P-D complexes compared to the wt U6 promoter probe (data not shown).

PBP

-

re-hTBP

++++++++++ -

-+++++

of the P-D-T complex

is dependent

1234567891011

Fig. 5. Detection

of the TATA box to the PSE in the U6 proximal

on the proximity

promoter

probes. The

wt and mutant probes used in EMSA (as described in Fig. 1) are noted above each lane. Lanes 2 to 6 contained 2 ug PBP added to the radiolabeled probes.

Lanes 7 to 11 contained

2 ug PBP and 0.08 ug re-hTBP

added to the radiolabeled probes. The band marked by an asterisk represents a non-specific complex. An open bracket indicates a smear whose

presence

containing

was dependent

on the addition

of TBP

to a probe

the TATA box.

This result was unexpected,

since transcription

from both

inP and deP mutant promoters was impaired when the spacing between the PSE and TATA box was altered by more than 2 bp (Goomer and Kunkel, 1992). It is interesting that formation of the P-D-T complex on the deP5 probe was not reduced, in contrast to the inP5 probe, since the relative helical orientation of the binding sites was markedly changed with both mutant probes. It appears as if the distance of the separation between the PSE and the TATA box is more important than the rotational orientation on the double helix. By comparing EMSA and DNaseI footprinting results of re-yTBP-DNA complexes, Hawley and coworkers have demonstrated that these protein-DNA complexes dissociate in gels (Hoopes et al., 1992). The lower intensity of the P-D-T complex formed on the inPl0 promoter may reflect either the disability of TBP to become incorporated into a P-D-T (i.e., a lower k,, for inP10) or the relative ease of dissociation of TBP from P-D-T (i.e., a higher koff for inP10). When binding reactions were incubated longer (up to 120 min) and the time of electrophoresis was unchanged, no additional complex formation could be detected (data nbt shown). When gels were electrophoresed longer, the amount of P-D-T complex

274 detected ished shorter

on both

wt and inPl0

significantly

probes

in comparison

was not dimin-

to that

seen

after

times (data not shown).

(1) A HE-binding

protein

on the human

of re-TBP

moter probe resulted taining both proteins (3) Formation on mutant

(P-D)

can

be

This complex

of polyclonal

Ab prepared

along with PBP to a U6 pro-

in the formation (P-D-T).

of the P-D-T

U6 promoters

ally and contain

complex

116 gene promoter.

was disrupted by the addition against re-TBP. (2) Addition

I transcription factor. SLI. Cell 6X ( 1992) 965 976. Cormack. B.P. and Struhl, K.: The TATA-binding protein for transcription by all three nuclear cells. Cell 69 ( 1992) 685 ~696.

(d) Conclusions detected

L.. TaneSc, N. and ~JI:LI~. R.. Ifhc TATA-binding ~I-V~CII~ and associated factors arc integral components of the RNA polymct-asc

C’Olnai,

complex

of a complex

con-

was diminished

that are defective transcription-

increased

spacing

between

the PSE and

TATA box.

Goomer, R.S. and Kunkcl. G.R.: The transcriptional human U6 small nuclear RNA gene is dictated

together,

the above

observations

indicate

that the protein factors that bind the PSE and the TATA box on the U6 proximal promoter interact with one another, either directly or indirectly. Since our PBP fraction was not homogeneous, these experiments cannot rule out the possibility of the involvement of other ‘bridge factors’ that may facilitate the apparent interaction between PBP and TBP. The ‘bridge factors’ could be stably bound PBP-associated factors or independent proteins that cofractionate with PBP in our preparations.

in yeast

start site for a by a compound

promoter element consisting of the PSE and the TATA box. Nucleic Acids Res. 20 ( 1992) 4903 4912. Hahn. S.. Buratowski. S.. Sharp. PA. and Guarente. L.: Isolation of the gene encoding the yeast TATA binding protein TFIID: a gene identical to the SPTIS

suppressor

of Ty element

insertions.

Cell 58

(1989)1173~11X1. Hernander, N.: TBP. a universal Dev. 7 (1993) 1291 130X. Hoopes,

eukaryotic

B.C., LeBIanc, J.F. and Hawley,

transcription

P.M.. Schmidt.

factor‘? Genes

D.K.: Kinetic analysis

TFIID-TATA box complex formation suggests way. J. Biol. Chem. 267 ( 1992) I1539- 11547. Kao. C.C., Lieberman.

(4) Taken

is required

RNA polymerases

of yeast

a multi-step

path-

M.C.. Zhou, Q.. Pei. R. and Berk,

A.J.: Cloning of a transcriptionally active factor. Science 248 ( 1990) 1646-- 1650.

human

TATA binding

Kassavetis. G.A., Joazeiro. C.A.P.. Pisano, M.. Geiduschek. E.P.. Colbert. T., Hahn, S. and Blanco. J.A.: The role of the TATA-binding protein in the assembly and function of the multisubunit yeast RNA polymerase 111 transcription factor. TFIIIB. Cell 71 (1992) 1055%1064. Kunkel, G.R. and Danzeiser,

D.A.: Formation

of a template

committed

complex on the promoter of a gene for the U6 small nuclear RNA from the human requires multiple sequence elements, including the distal region. J. Biol. Chem. 267 (1992) 14250~~14258. Kunkel. G.R. and Pederson,

T.: Upstream

elements required

for efficient

transcription of a human U6 RNA gene resemble those of Ul and U2 genes even though a different polymerase is used. Genes Dev. 2 ACKNOWLEDGEMENTS

We would like to thank Tjian’s laboratory (U.C. Berkeley) for the hTBP cDNA, Kathy Beifuss from the David 0. Peterson laboratory (Texas A&M) for TBP preparations, Rachel Meyers and Phillip Sharp (MIT) for rabbit laboratory,

a-TBP Ab and Stephan Kopytek (Peterson Texas A&M) for rabbit a-TFIIB Ab. We are

grateful for the expert assistance of Deborah A. Danzeiser with cell culture and protein purification, and JungSun Park for subcloning the hTBP cDNA into a pET3c vector. This work was supported by grants from the NSF (DMB-8903970 and MCB-9304799).

REFERENCES Ares Jr., M., Mangin, M. and Weiner, A.M.: Orientation-dependent transcriptional activator upstream from a human U2 snRNA gene. Mol. Cell. Biol. 5 (1985) 1560-1570. Bernues, J., Simmen, K.A., Lewis, J.D., Gunderson, S.I., PolycarpouSchwarz. M., Moncollin, V., Egly, J.-M. and Mattaj, I.W.: Common and unique transcription factor requirements of human Ul and U6 snRNA genes. EMBO J. 12 (1993) 3573-3585. Ciliberto, G., Buckland, R., Cortese, R. and Philipson, L.: Transcription signals in embryonic Xenopus la& Ul RNA genes. EMBO J. 4 (1985) 1537-1543.

(1988) 196-204. Lobo, S.M. and Hernandez.

N.: A 7 bp mutation

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