Three key subregions contribute to the function of the downstream RNA polymerase II core promoter

Abstract

The RNA polymerase II core promoter is a diverse and complex regulatory element. To gain a better understanding of the core promoter, we examined the motif 10 element (MTE), which is located downstream of the transcription start site and acts in conjunction with the initiator (Inr). We found that the MTE promotes the binding of purified TFIID to the core promoter and that the TAF6 and TAF9 subunits of TFIID appear to be in close proximity to the MTE. To identify the specific nucleotides that contribute to MTE activity, we performed a detailed mutational analysis and determined a functional MTE consensus sequence. These studies identified favored as well as disfavored nucleotides and demonstrated the previously unrecognized importance of nucleotides in the subregion of nucleotides 27 to 29 (+27 to + 29 relative to A(+1) in the Inr consensus) for MTE function. Further analysis led to the identification of three downstream subregions (nucleotides 18 to 22, 27 to 29, and 30 to 33) that contribute to core promoter activity. The three binary combinations of these subregions lead to the MTE (nucleotides 18 to 22 and 27 to 29), a downstream core promoter element (nucleotides 27 to 29 and 30 to 33), and a novel "bridge" core promoter motif (nucleotides 18 to 22 and 30 to 33). These studies have thus revealed a tripartite organization of key subregions in the downstream core promoter.

FIG. 1.

Purification of TFIID from Drosophila S2 cells containing FLAG-tagged TBP. (A) Synthesis of FLAG-tagged TBP in S2 cells. Whole-cell lysates derived from S2 cells and two different FLAG-TBP-containing S2 cell lines were subjected to Western blot analysis with antibodies against Drosophila TBP. (B) Scheme for the purification of TFIID from S2 cells containing FLAG-tagged TBP. (C) Purification of TFIID containing FLAG-tagged TBP. The polypeptides were resolved by 10% polyacrylamide-SDS gel electrophoresis and visualized by silver staining. In addition, by Western blotting and mass spectrometry, the purified TFIID was found to contain FLAG-tagged TBP as well as all TAFs from TAF1 through TAF14.

FIG. 2.

The MTE contributes to the binding of purified TFIID to the core promoter. The wild-type, m18-22 (MTE-inactivating), m30-33 (DPE-inactivating), and double mutant (m18-22 and m30-33) versions of the Drosophila Tollo (A) and CG10479 (B) core promoters were subjected to DNase I footprinting analysis with purified Drosophila TFIID. The positions of the Inr (−2 to + 4, relative to the position defined as A₊₁ in the Inr consensus), MTE (depicted as sequences from +18 to +27), and DPE (+28 to +33) motifs are indicated. In the wild-type promoters, regions of DNase I protection and hypersensitivity are indicated by brackets and filled dots, respectively.

FIG. 3.

The addition of an MTE increases the affinity of TFIID for the core promoter. The wild-type (WT) and WT+MTE (containing the Tollo MTE sequence from +18 to +27, relative to the A₊₁ position in the Inr) versions of the Drosophila E74B (A) and Doc (B) core promoters were subjected to DNase I footprinting analysis with purified Drosophila TFIID. The positions of the Inr (−2 to +4), MTE (Tollo core promoter sequence from +18 to +27), and DPE (+28 to +33) motifs are indicated. In the WT+MTE promoters, regions of DNase I protection and hypersensitivity are indicated by brackets and filled dots, respectively.

FIG. 4.

The TAF6 and TAF9 subunits of TFIID appear to be in close proximity to the MTE. (A) Diagram of photoaffinity probes containing AB-dUMP at the +20, +25, or +30 position relative to the +1 transcription start site. The Tollo core promoter sequences are shown. The diagram is roughly to scale. (B) Photo-cross-linking of purified TFIID with the Tollo core promoter. Reactions were performed in the presence or absence of TFIID as well as with or without UV irradiation, as indicated. (C) Photo-cross-linking of purified TFIID with the E74B core promoter containing the Tollo MTE. The E74B+MTE core promoter is identical to the construct that was used in the DNase I footprinting analysis shown in Fig. 3A. Reactions were performed as described for panel B.

FIG. 5.

Single-nucleotide substitution analysis reveals sequences that are important for MTE activity. (A and C) Single-nucleotide substitution analysis of the MTE in the Drosophila Tollo (A) and CG10479 (C) core promoters. Mutant promoters containing every possible single-nucleotide substitution from +15 to +29 (relative to the A₊₁ in the Inr) were generated. To eliminate the contribution of the DPE in these experiments, all of the constructs contain the m30-33 mutation (CATA from +30 to +33), which inactivates DPE function. The promoters were subjected to in vitro transcription analysis with a Drosophila embryo nuclear extract, and the resulting transcripts were detected by primer extension-reverse transcription analysis. The primer extension data that correspond to the wild-type (WT) promoter are boxed. (B and D) The relative transcriptional activities of wild-type and mutant MTE sequences in the Tollo (B) and CG10479 (D) core promoters. Quantitation of the data from three independent experiments is shown. The data are normalized to promoters containing the wild-type MTE sequence. The error bars represent the standard deviations.

FIG. 6.

Identification of sequences that are important for MTE activity. The data shown in Fig. 5 were analyzed as follows. For each position, nucleotides that resulted in >90% of the transcriptional activity of the wild-type nucleotide (defined to be 100%) for both the Tollo and CG10479 promoters were designated favored nucleotides, whereas nucleotides that resulted in <60% of the transcriptional activity of the wild-type nucleotide for both promoters were designated disfavored nucleotides. The computationally derived motif 10 sequence (19) and the MTE sequence based on the initial characterization of the element (18) are also shown.

FIG. 7.

Analysis of three downstream subregions that are important for MTE and DPE activity. (A) Diagram of the Inr and downstream core promoter subregions. The relative locations of the elements are drawn approximately to scale. (B) Systematic analysis of three downstream subregions of the wild-type Tollo core promoter. A set of promoters with all possible combinations of the m18-22, m27-29, and m30-33 mutations in the Tollo core promoter was constructed. For each promoter, the wild-type (+) and mutant (−) versions of each sequence are indicated. The numbers below each promoter construct are the mean of three or four independent experiments relative to the wild-type Tollo promoter. (C and D) Analysis of three downstream subregions in E74B- and Doc-based core promoters. The relative contributions of the 18-22, 27-29, and 30-33 subregion sequences were tested by using the hybrid E74B+MTE and Doc+MTE core promoters, which consist of the natural E74B and Doc promoters containing the Tollo MTE sequences from +18 to +27 relative to the A₊₁ in the Inr. Thus, the hybrid promoters contain Inr, MTE, and DPE motifs. Beginning with each hybrid promoter, a set of promoters that comprises all possible combinations of the m18-22, m27-29, and m30-33 mutations was constructed. For each promoter, the wild-type (+) and mutant (−) versions of each sequence are indicated. The numbers below each promoter construct indicate the mean of three or four independent experiments relative to the hybrid core promoter containing the optimal sequences in the Inr, MTE, and DPE motifs.

FIG. 8.

Natural core promoters that are driven predominantly by the MTE or bridge core promoter motifs. (A and B) Natural TATA-less, MTE-containing core promoters that lack a strong DPE motif. Wild-type and mutant versions of the CG5397 (A) and CG6980 (B) core promoters were subjected to in vitro transcription and primer extension analyses. In addition to the standard m18-22 and m30-33 mutations, we analyzed promoters in which a consensus DPE sequence was introduced from +30 to +33 (Consensus 30-33). The relative activity values are the means of three or four independent experiments normalized to the cognate wild-type promoter. (C) Natural TATA-less, bridge (subregion 18-22 plus 30-33)-containing core promoter that lacks a strong sequence at nucleotides +27 to +29. Wild-type and mutant versions of the CG15253 core promoter were subjected to in vitro transcription and primer extension analyses. In addition to the m18-22, m27-29, and m30-33 mutations, we tested a mutant promoter containing an optimal sequence at positions 27 to 29 (Optimal 27-29) (Fig. 6). The relative activity values are the means of three or four independent experiments normalized to the value of the cognate wild-type promoter.

FIG. 9.

Model of a tripartite organization of key interaction points of TFIID with downstream core promoter sequences. In this model, the 18-22, 27-29, and 30-33 subregion sequences are three key downstream points of interaction of TFIID with the core promoter. Based on the photo-cross-linking studies (Fig. 4), the TAF6 and TAF9 subunits of TFIID are shown in close proximity to the downstream core promoter region. The Inr is included because it has been previously shown that the MTE as well as the DPE acts in a cooperative manner with the Inr (4, 18). The MTE comprises the 18-22 and 27-29 subregion sequences, whereas the DPE contains the 27-29 and 30-33 subregion sequences. In addition, the 18-22 and 30-33 subregion sequences can function synergistically in the absence of an optimal 27-29 sequence and form the bridge element.