PROTEIN

EXPRESSION

3, 374-379

AND PURIFICATION

(19%)

Expression in Escherichia co/i: Purification and Properties of the Recombinant Human General Transcription Factor rTFllB Vincent

Moncollin,

Laboratoire l’INSERM,

Received

Laurent

Schaeffer,

Christian

Chalut,

February

7, 1992,

and

in revised

form

May

Eglyl

23, 1992

The human class II transcription factor TFIIB (rTFIIB) was overexpressed in Escherichiu coli using a T7 RNA polymerase expression system and further purified to apparent homogeneity. The purified rTFIIB is identical to the endogeneous factor according to the following criteria: molecular weight, microsequencing and mass spectra studies, ability to recognize the stable preinitiation complex formed between TFIID and the adenovirus 2 major late TATA box as demonstrated by gel shift as well as by DNase I footprinting assays, and transcription activity. 0 1992 Academic Press, 1~.

The transcription factor TFIIB is one of several general transcription factors [such as TFIIA/STF, TFIID, TFIIE, TFIIF/RAP30-RAP74, BTF2, TFIIG, and TFIIH; for reviews see Refs. (l-3)] required by RNA polymerase B (II) for accurate transcription initiation from eukaryotic minimal promoters (defined as the region including the TATA box and the transcription start site). Transcription factor TFIIB was recently purified (4-6) and subsequently cloned (5). The nucleotide sequence of human TFIIB has an open reading frame encoding a polypeptide of 316 amino acids with a calculated molecular mass (M,) of 34,832, which is in agreement with the apparent molecular weight of TFIIB estimated by SDS-PAGE, gel filtration, and/or glycerol gradient centrifugation ($6). TFIIB has a basic character (p1- 8.32) and presents some sequence similarities with the u factor (5). TFIIB, an absolutely required general transcription factor, does not by itself recognize specifically the minimal promoter sequence. However, it is postulated that TFIIB is drawn into the transcription ’ To whom 374

and Jean-Marc

de Ge’ne’tique Mole’culaire des Eucaryotes du CNRS, U/184 de Biologie Mol&ulaire et de GEnie Ge’ne’tique de Institut de Chimie Biologique, Faculte’ de Mkdecine, 11 rue Humann, 67085 Strasbourg Cedex, France

correspondence

should

be addressed.

initiation mechanism through an association with the so-called DA complex. Indeed, this complex, which results from an interaction between TFIID, TFIIA/STF, and the promoter (7), is recognized by TFIIB as observed by gel retention and DNase I footprinting assays (5,6). It has been postulated that the association of TFIIB with the DA complex can be facilitated by some activators such as GAL4-VP16 (8). In order to study the structure-function relationship by crystallography, on the one hand, and to further screen the various cellular extracts to identify the other components involved in the transcription mechanism, it is important that large quantities of pure and biologically active transcription factor be available. As previously reported for yeast TFIID factor (9), we present here the expression and the purification of the soluble recombinant TFIIB (rTFIIB) protein and the subsequent comparison of some of its physical and biochemical characteristics with those of the endogeneous material. MATERIALS

Preparation

AND

METHODS

of Soluble rTFIIB

Extracts

A starter culture of 20 ml LB containing ampicillin (100 pug/ml) was inoculated with Escherichia coli strain BL21 (DE3) phIIB [Ref. (5); a generous gift from D. Reinberg] and grown overnight at 37°C. This starter was used to inoculate 2 liters LB medium containing ampicillin (100 pg/ml). Cultures were grown at 37°C to an ODsoo of 0.6 to 1.0 and then induced by addition of IPTG to 0.5 mM. After a further 2 h, cells were harvested by centrifugation and resuspended in 100 ml of buffer A (50 mM Tris-HCl, pH 7.9, 0.1 mM EDTA, 0.5 mM DTT, 20% glycerol, and 100 mM KCl). The cells were lysed by freeze-thawing once in liquid nitrogen 1046-5928192

$5.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

TRANSCRIPTION

FACTOR

and cold water, respectively, and sonication for 4 X 30 s at power 8,70% cycle (Vibracell, Bioblock Scientific), in 2 X 50-ml aliquots. The extract was clarified by centrifugation for 15 min at 6000g in a Beckman R45 Ti rotor. rTFIIB

Purification

Fifty’milliliters of the loo-ml extract, prepared as described above, was loaded onto a 50-ml phosphocellulose (Whatman) column (2.5 cm i.d. X 10 cm; flow rate, 40 ml/h) equilibrated in buffer A. The column was then sequentially washed with 100 ml of buffer A, 100 ml of buffer A plus 0.3 M KCl, and 100 ml of buffer A plus 0.5 M KCl. Proteins eluted during the 0.5 M KC1 step were collected in lo-ml fractions; the fractions containing rTFIIB (fractions 2-4 as judged by SDS-PAGE; 30 ml) were pooled and dialyzed for 4 h against 1 liter of buffer A. Ten milliliters of the sample was then loaded onto an 8-ml Sulfopropyl Fast Flow (Pharmacia, Uppsala, Sweden) column (1 cm i.d. X 10.2 cm; flow rate, 15 ml/h) equilibrated in buffer A. After the column was washed with 10 ml of buffer A and 16 ml of buffer A + 0.2 M KCl, the bound proteins were eluted with a 20-ml linear 0.2 to 0.7 M KC1 gradient in 80 min and l-ml fractions were collected along the gradient. The fractions containing rTFIIB (fractions 14-19 as judged by SDS-PAGE; 6 ml) were pooled and dialyzed for 2 h against 1 liter of buffer A + 0.05 M KCl; 6 ml of the sample was then loaded onto an analytical DEAE-5PW (Toso-Haas, Stuttgart, Germany) column (0.75 cm i.d. X 7.5 cm; flow rate, 0.6 ml/ min) equilibrated in buffer A + 0.05 M KCl. After the column was washed with 10 ml of buffer A + 0.05 M KCl, the bound proteins were eluted with a 12-ml linear 0.05 to 0.40 M KC1 gradient run in 20 min and 0.6-ml fractions were collected along the gradient. The rTFIIB is eluted at 0.15 M KCl. In Vitro Runoff Transcription

Assay

The in uitro transcription system was as previously described (6) and was reconstituted for assaying TFIIB activity with the following fractions as sources of general transcription factors: 5 ~1 of DE 0.20, containing BTF3 and BTF4 (TFIIF) [Refs. (6,lO) and C. Chalut unpublished data], 5 ~1 of DElM containing PolII and TFIIE, 3 ~1 of SP 0.3 containing BTF2 (ll), 1 ~1 of DE0.35 containing TFIIASTF (12), and0.2 ~1of recombinant human TFIID expressed in E. coli (9,13-15). The template used was the EcoRI-Sal1 fragment of pM34 (648 bp long), which includes adenovirus 2 major late promoter (Ad2MLP) sequences from -34 to +33 [(16); 309 nucleotide-long runoff transcript]. Polyacrylamide

Gel Electrophoresis

and Western Blotting

SDS-PAGE was performed on 12% gels under reducing conditions. When migration was complete the gels

rTFIIB

375

PROPERTIES

were either stained (Coomassie blue R-250 or silver) or Western blotted as follows. The gels were rinsed for a few seconds in the electroblotting buffer (10 mM Trisbase, 75 mM glycine, 0.4% methanol) and then sandwiched between nitrocellulose filters and several sheets of Whatman 3MM paper. Electroblotting was performed at constant voltage (90 V) for 1 h in the electroblotting buffer; filters were washed briefly in 1X phosphate-buffered saline (PBS), blocked by incubating for 1 h at 25°C in PBS containing 3% milk powder, and then incubated for 1 h at 37°C with the antibody in PBS containing 0.3% milk powder. The antibody used was raised against the peptide (one-letter code) TDFKFDTPVDKLPQL, which corresponds to the COOH-terminal sequence of the TFIIB protein (5). The filters were washed in 0.05% Tween 20, incubated 1 h at 25°C with a peroxydase-coupled antibody (goat anti-rabbit; Jackson Immuno Research-Interchim), washed in 0.05% Tween 20, dried, and revealed by the enhanced chemioluminescence method (Amersham, UK) and autoradiography. DNase I Footprinting

and Gel Retention

Assays

DNase I footprinting consisted of a 30-min preincubation at 30°C in a 20-~1 reaction volume containing variable amounts of the protein fraction, - 1 ng (10,000 cpm) of the DNA fragment pM7 SmaI-BamHI [-54 to +33 of the Ad2MLP sequences; Ref. (16)] 5’-end-labeled at the BamHI site, 100 ng of poly(dG-dC)(dG-dC), 0.05% NP-40,5 mM MgCl,, and 50 pg/ml BSA in buffer A containing 50 mM KCl. After the preincubation, 2 ~1 of DNase I (10 pglml), (Worthington, NJ) was added for 2 min at 30°C and reactions were stopped by adding 0.4 ml of a solution containing 0.5% sodium dodecyl sulfate, 50 mM sodium acetate (pH 5), and 50 pg/ml E. coli tRNA. DNA digestion products were analyzed on 8% acrylamide-8 M urea gels as described previously (6). The gel retention assay consisted of a preincubation step identical to that of the footprinting assay in a lo-411 reaction volume containing 250 ng of poly( dG-dC) (dGdC!) as a nonspecific DNA competitor and 0.1~1 of STF/ TFIIA (12). After 30 min at 30°C 1.5 ~1 of 87% glycerol was added and the mixture electrophoresed at 30 mA on a 4.5% polyacrylamide gel (ratio acrylamide/bisacrylamide: 80/l) until bromophenol blue, run in parallel, reached the bottom of the gel. The electrophoresis buffer was 25 mM Tris-base, 190 mM glycine, 1 mM EDTA, pH 8.5. The gel was dried and autoradiographed. RESULTS

rTFIIB

Purification

rTFIIB activity was monitored both by Western blot using polyclonal antibodies and by in vitro transcription of a template containing the Ad2MLP [pM34; Refs. (6,16) and Materials and Methods]. The purification

376

MONCOLLIN

I

FTO.1

I

I

I

0.2-0.70MKCI

0.05

Expression in Escherichia coli: purification and properties of the recombinant human general transcription factor rTFIIB.

The human class II transcription factor TFIIB (rTFIIB) was overexpressed in Escherichia coli using a T7 RNA polymerase expression system and further p...
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