Structural and functional interactions of transcription factor (TF) IIA with TFIIE and TFIIF in transcription initiation by RNA polymerase II

Abstract

A topological model for transcription initiation by RNA polymerase II (RNAPII) has recently been proposed. This model stipulates that wrapping of the promoter DNA around RNAPII and the general initiation factors TBP, TFIIB, TFIIE, TFIIF and TFIIH induces a torsional strain in the DNA double helix that facilitates strand separation and open complex formation. In this report, we show that TFIIA, a factor previously shown to both stimulate basal transcription and have co-activator functions, is located near the cross-point of the DNA loop where it can interact with TBP, TFIIE56, TFIIE34, and the RNAPII-associated protein (RAP) 74. In addition, we demonstrate that TFIIA can stimulate basal transcription by stimulating the functions of both TFIIE34 and RAP74 during the initiation step of the transcription reaction. These results provide novel insights into mechanisms of TFIIA function.

Fig. 1. Photo-cross-linking of rTFIIA on the AdMLP

A, photo-cross-linking experiments with photoprobes −39/−40 and +26 were performed in the presence of rTFIIA (α/β and γ), TBP, TFIIB, TFIIF (RAP74 and RAP30), TFIIE (p56 and p34), and RNAPII. The specificity of the cross-linking signals was assessed by comparing reactions performed with all the factors to ones performed in the absence of TBP. Under our reaction conditions, the absence of TBP has the same effect as the use of a photoprobe with a mutation in the TATA element (TATAAAA to TAGAGAA) (55). The position of TFIIA α/β and TFIIA γ are indicated. B, schematic representation of promoter contacts by TFIIA in the context of the TBP-TFIIA-promoter complex and the TBP-TFIIA-TFIIB-TFIIF-RNAPII-TFIIE-promoter complex in which the DNA adopts a wrapped structure. Positions −39/−40, +26 and +1 are indicated. Only TBP, TFIIA, and RNAPII are represented to simplify the diagram.

Fig. 2. Interactions of natural TFIIA with components of the basal transcription machinery

Protein-affinity chromatography was performed using microcolumns containing immobilized RNAPII, RAP74, RAP30, TFIIE56, TFIIE34, TFIIB, TBP, and BSA. A volume of 50 μl of nTFIIA (100 ng of α) was chromatographed through each column. The flowthrough was collected in each case. The columns were eluted with 50 μl of buffer containing increasing amounts of NaCl (e.g. 0.1, 0.3, and 0.5 M). The fractions were analyzed using SDS-PAGE and compared with the input (I). The positions of the nTFIIA subunits and a contaminant polypeptide of the TFIIA preparation that served as negative control are indicated. In each case, a diagram showing the relative intensities of both TFIIAα and a contaminant band (negative control) in the various fractions is shown.

Fig. 3. Interactions of TFIIAα/β and TFIIAγ with RAP74, TFIIE56, and TFIIE34

Protein-affinity chromatography was performed using microcolumns containing immobilized RAP74, TFIIE34, and TFIIE56. A volume of 50 μl containing 200 ng of TFIIAα/β and TFIIAγ was chromatographed through each column. Fractions were collected and analyzed as in Fig. 2. The positions of α/β, γ, and BSA (as an internal negative control) are indicated.

Fig. 4. Interactions of TFIIAα/β with RAP74 deletion mutants

A, protein affinity chromatography was performed using columns containing immobilized RAP74 fragments (wild type or deletions mutants). A volume of 50 μl containing 200 ng of TFIIAα/β was chromatographed through each column. Fractions were collected and analyzed as in Fig. 2. The positions of TFIIAα/β and BSA (as an internal negative control) are indicated. B, summary of the interactions between the RAP74 deletion mutants and TFIIA. The various domains of RAP74 are indicated in the top part. The two TFIIA-binding domains of RAP74 are deduced from our analysis.

Fig. 5. Association of RAP74 with a TBP-TFIIA-promoter complex

Gel mobility shift assays were performed using a radiolabeled DNA fragment comprising the AdMLP in the presence of TBP alone, TBP, and TFIIA and TBP, TFIIA and various fragments of RAP74 (RAP74-(1–517;wt), RAP74-(1–75), RAP74-(1–136), RAP74-(363–409), and RAP74-(363–444)). The positions of the TBP (T), TBP-TFIIA (T-A), and TBP-TFIIA-RAP74 (T-A-RAP74) complexes and that of the free probe are indicated.

Fig. 6. Stimulation of basal transcription by TFIIA

A, run-off transcription assays were performed on a supercoiled template carrying the AdMLP using TBP, TFIIB, TFIIE, TFIIF, and RNAPII in either the absence or the presence of increasing amounts of TFIIA (100, 200, and 400 ng). The position of the accurately initiated transcript (391 nt) is indicated. B, abortive initiation assays were performed on a synthetic double-stranded oligonucleotide carrying the AdMLP using TBP, TFIIB, RAP30, and RNAPII in either the absence or the presence of RAP74 alone, RAP74 and TFIIE34, and RAP74 and TFIIE56. Increasing amounts of TFIIA were added to the reactions. The positions of the abortive transcripts (4–10 nt) are indicated. C, quantification of the stimulatory effect of TFIIA on abortive initiation. The intensity of the bands corresponding to the abortive transcripts from 4–6 experiments were quantitated using a PhosphorImager. The measured intensities were used to calculate the ratios of amount of transcript produced in the presence of TFIIA to that produced in its absence in each case (Fold Stimulation).