Stimulation of the XPB ATP-dependent helicase by the beta subunit of TFIIE

Abstract

TFIIE and TFIIH are essential for the promoter opening and escape that occurs as RNA polymerase II transits into early elongation. XPB, a subunit of TFIIH, contains an ATP-dependent helicase activity that is used in both of these processes. Here, we show that the smaller beta subunit of TFIIE stimulates the XPB helicase and ATPase activities. The larger alpha subunit can use its known inhibitory activity to moderate the stimulation by the beta subunit. Regions of TFIIE beta required for the helicase stimulation were identified. Mutants were constructed that are defective in stimulating the XPB helicase but still allow intact TFIIE to bind and recruit XPB and TFIIH to form the pre-initiation complex. In a test for the functional significance of the stimulatory effect of TFIIE beta, these mutant forms of TFIIE were shown to be defective in a transcription assay on linear DNA. The data suggest that the beta subunit of TFIIE is an ATPase and helicase co-factor that can assist the XPB subunit of TFIIH during transcription initiation and the transition to early elongation, enhancing the potential diversity of regulatory targets.

Figure 1

TFIIEβ stimulates XPB helicase activity. (A) Helicase assay. Lane 1, substrate alone; lane 2, substrate heated for 2 min to unanneal radioactively labeled oligonucleotide; lane 3, 0.8 μg TFIIEβ was added; lane 4, 2.4 μg of XPB was added; lanes 5 and 6, 0.4 and 0.8 μg of TFIIE were added to 2.4 μg of XPB, respectively. (B) A graphical representation of lane 4–6 in (A). (C) Helicase assay. Lane 1, substrate alone; lane 2, 0.8 μg of TFIIEβ and 2.4 μg of GST were added; lane 3, 0.8 μg of TFIIEβ and 11 μg of GST were added; lane 4, 0.8 μg of TFIIEβ were added to 2.4 μg of XPB. (D) ATPase assay. Lane 1, ATP alone; lane 2, 0.13 μg of TFIIEβ was added; lane 3, 0.8 μg of XPB was added; lane 4 contained both 0.13 μg of TFIIEβ and 0.8 μg of XPB. The ATPase stimulation was 1.8 ± 0.4-fold.

Figure 2

The C-terminus of TFIIEβ is important for the stimulation of XPB helicase activity. (A) A diagram of TFIIEβ functional domains (36) and the C-terminal truncation mutants of IIEβ were depicted. (B) Helicase assay, conditions as in Figure 1A. Lane 1, substrate alone; lane 2, substrate heated for 2 min; lane 3, XPB was added; lane 4, as in lane 3, except wild-type TFIIEβ was added; lane 5, equimolar of Δ257–291 as in lane 4 TFIIEβ was added to XPB; lane 6, Δ197–291 was added to XPB; lane 7, Δ149–291 was added to XPB. (C) A graphical representation of XPB helicase stimulation by wild-type and various TFIIEβ mutants.

Figure 3

TFIIEβ stimulates TFIIH helicase and ATPase activity. (A) Helicase assay. Lane 1, XPB 3′–5′ helicase specific substrate; lane 2, substrate was heated for 1 min; lane 3, 5 and 7 contained 30 ng of TFIIH and the reaction times were 15, 30 and 45 min, respectively; lane 4, 6 and 8 contained 80 ng of TFIIEβ and 30 ng of TFIIH and the reaction times are 15, 30 and 45 min, respectively. (B) ATPase assay. Lane 1, ATP alone; lane 2, 80 ng of TFIIEβ was added; lane 3, 60 ng of TFIIH was added; lane 4 contained 80 ng of TFIIEβ and 60 ng of TFIIH. The ATPase stimulation was 2.2 ± 0.4-fold.

Figure 4

TFIIEα prevents TFIIEβ from stimulating XPB. (A) Helicase assay. Lane 1, substrate alone; lane 2, XPB was added; lane 3, 0.8 μg of TFIIEβ and XPB were included. For lanes 4 and 5, 1.6 and 3.2 μg, respectively, of IIEα were incubated with 0.8 μg of TFIIEβ for 15 min before the addition of XPB and the substrate. (B) A graphical representation of the helicase activities of samples in lanes 3–5.

Figure 5

TFIIEα binds to XPB to counteract TFIIEβ stimulation. (A) Western blot of wild-type TFIIEα and its mutants. (B) CoIP of TFIIEβ with wild-type TFIIEα and its mutants. (C) Helicase assay. Lane 3, XPB was added; lane 4, TFIIEβ and XPB were added (70% of substrate was unwound); lanes 5 and 6 as in lane 4, except 2.4 μg of TFIIEα or 50–439 were added, respectively (∼30% of substrate were unwound for both).

Figure 6

Mutants that fail to stimulate XPB helicase activity also have transcription defects. (A) In vitro transcription at the AdML promoter. Lane 1, TFIIE depleted extract; lane 2, complementation of wild-type TFIIEβ and TFIIEα; lane 3, with mutant I171S and TFIIEα (∼0% transcription activity compared with wild-type); lane 4, with mutant I189S and TFIIEα (∼60% transcription activity compared with wild-type). (B) Helicase assay. Lane 1, 2 μg of XPB; lane 2, XPB and 1.2 μg of wild-type TFIIEβ (3-fold stimulation); lane 3, XPB and 1.2 μg of I171S; lane 4, XPB and 1.2 μg of I189S. Only the released oligo is shown. (C) CoIP with TFIIEβ and general transcription factors. Top panel: lane 1, anti-FLAG resin plus wild-type TFIIEβ; lanes 2–4 anti-FLAG resin pre-bound with FLAG-tagged TFIIEα plus wild-type TFIIE or its mutants. Middle panel: lane 1, glutathione resin with GST plus wild-type TFIIEβ; lanes 2–4 glutathione resin with GST–XPB plus wild-type TFIIEβ or its mutants. Bottom panel: lane 1, anti-FLAG resin plus wild-type TFIIEβ with His₆ tag; lanes 2–4, anti-FLAG resin pre-bound with FLAG-tagged wild type TFIIEβ 6× His tag or its mutants. (D) Pre-initiation complex formation of wild-type TFIIEβ and its mutants. Lane 1, beads with equimolar of the template without the E4 promoter and gal sites are incubated with TFIIEβ depleted extract complement with recombinant wild-type TFIIE. Lanes 2–4, beads bound to G9E4 template are incubated with recombinant wild-type TFIIE, I171S or I189S, respectively. The left panel indicates the transcription factors that are being probed. (E) In vitro transcription at the G9E4 promoter. Lane 1, TFIIE depleted extract; lane 2, complementation of wild-type TFIIE; lane 3, with mutant I171S and TFIIEα; lane 4, with mutant I189S and TFIIEα.