The archaeal TFIIEalpha homologue facilitates transcription initiation by enhancing TATA-box recognition

Abstract

Transcription from many archaeal promoters can be reconstituted in vitro using recombinant TATA-box binding protein (TBP) and transcription factor B (TFB)--homologues of eukaryal TBP and TFIIB--together with purified RNA polymerase (RNAP). However, all archaeal genomes sequenced to date reveal the presence of TFE, a homologue of the alpha-subunit of the eukaryal general transcription factor, TFIIE. We show that, while TFE is not absolutely required for transcription in the reconstituted in vitro system, it nonetheless plays a stimulatory role on some promoters and under certain conditions. Mutagenesis of the TATA box or reduction of TBP concentration in transcription reactions sensitizes a promoter to TFE addition. Conversely, saturating reactions with TBP de-sensitizes promoters to TFE. These results suggest that TFE facilitates or stabilizes interactions between TBP and the TATA box.

Fig. 1. Archaea possess a sequence homologue of the α-subunit of TFIIE. (A) An alignment of various archaeal TFE and eukaryal TFIIEa subunits is shown. Pyrab, Pyrococcus abyssi, DDBJ/EMBL/GenBank accession No. C75055; Pyrho, Pyrococcus horikoshii, accession No. B71106; Metja, Methanococcus jannaschii, accession No. Q58187; Metth, Methanobacterium thermoautrophicum, accession No. A69090; Halsp, Halobacterium sp. NRC-1, accession No. AAG19231; Arcfu, Archaeglobus fulgidus, accession No. E69344; Sulso, Sulfolobus solfatraicus, http://niji.imb.nrc.ca/sulfolobus; Aerpe, Aeropyrum pernix, accession No. F72503; human, accession No. P29083; yeast, accession No. P36100. Identical residues are boxed in black and homologous residues are shaded in grey. A helix–turn–helix structure is indicated by black horizontal bars. (B) Schematic representation of motifs found in archaeal TFE. (C) Coomassie blue-stained SDS–polyacrylamide gel containing 3 µg of purified recombinant TFE.

Fig. 2. TFE interacts with RNAP and TBP. (A) Ni-NTA pull-down assays were performed with 10 µg of S. solfataricus extract, as detailed in Methods, in the presence (TFE beads) or absence (control beads) of 100 ng recombinant TFE. The proteins pulled-down in the assay were detected by western blotting with antisera raised against TBP, TFB or RNAP B-subunit (Qureshi et al., 1997). Lanes contain 5% of input (In), unbound material (Un), wash (W) or material eluted from the beads (El). (B) Assays performed as above but with purified TBP and RNAP in place of extract.

Fig. 3. TFE stimulates transcription from some promoters. (A) In vitro transcriptions were carried out with the indicated promoters in reactions containing 20 ng TBP, 25 ng TFB and 200 ng RNAP for 10 min at 65°C. RNA products were detected by primer extension. (B) Sequence of TATA box and BRE of the promoters used in (A).

Fig. 4. TFE stimulates transcription under sub-optimal TBP–TATA-box interactions. (A) The sequence of wild-type and mutant T6 promoters is shown. The TATA box and BRE are boxed and the positions of sequence substitutions shown in bold and underlined. (B) The products of in vitro transcription of the promoters shown in (A) were detected by primer extension analysis and electrophoresed on a 8% denaturing polyacrylamide gel. (C) In vitro transcription reactions were performed on the 5S promoter, either with no TFE or supplemented with 50 ng of TFE as indicated. The reactions contained 25 ng TFB, 200 ng of RNAP and varying amounts of TBP as indicated. (D) In vitro transcription reactions were performed on the T6 promoter, either with no TFE or supplemented with 50 ng of TFE as indicated. Reaction conditions were as (C). (E) In vitro transcription assays performed on the T6 promoter. Reactions contained 20 ng TBP, 200 ng RNAP and either 40, 20, 10, 5 or 2.5 ng TFB.

Fig. 5. TFE stimulates TBP–TFB–DNA complex formation. DNase I footprinting analysis was performed on the T6 promoter in the presence or absence of 25 ng TFE and 20 ng TFBc (TFB core domain) as indicated. TBP was present at 20, 15, 10 or 5 ng per reaction (lanes 4–7 and 8–11).