Promoter scanning for transcription inhibition with DNA-binding polyamides

Abstract

When targeted to sequences adjacent to a TATA element, pyrrole-imidazole (Py-Im) polyamides inhibit the DNA binding activity of TATA box binding protein (TBP) and basal transcription by RNA polymerase II. In the present study, we scanned the human immunodeficiency virus type 1 promoter for polyamide inhibition of TBP binding and transcription using a series of DNA constructs in which a polyamide binding site was placed at various distances from the TATA box. Polyamide interference with either TBP-DNA or TFIID-TFIIA-DNA contacts both upstream and downstream of the TATA element resulted in inhibition of transcription. Our results define important protein-DNA interactions outside of the TATA element and suggest that transcription inhibition of selected gene promoters can be achieved with polyamides that target unique sequences within these promoters at a distance from the TATA element. Our studies also demonstrate the utility of the Py-Im polyamides for discovery of functionally important protein-DNA contacts involved in transcription.

FIG. 1.

Polyamide structures and binding sites. (A) Structures for polyamides ImPy-β-ImPy-γ-ImPy-β-ImPy-β-Dp (1), ImIm-β-ImIm-γ-PyPy-β-PyPy-β-Dp (2), and ImPy-β-PyIm-daba-PyIm-β-ImPy-β-Dp (3). Base sequence specificity depends on side-by-side pairing of Py and Im amino acids in the minor groove of DNA (6). Im-Py targets G·C base pairs and Py-Im targets C·G base pairs. Py-Py is degenerate and targets both A·T and T·A base pairs. The β-alanine-β-alanine pair also recognizes both A·T and T·A base pairs (42). Black and white circles, Im and Py rings, respectively; curved line, hairpin junction, which is formed with γ-aminobutyric acid or daba; diamonds, β-alanine; parenthesis with plus sign, Dp. The positive charge on the daba turn amino acid (3) is not shown. Me, methyl. (B) DNA sequences of the HIV-1 promoter constructs (from nucleotide positions −55 to +1, with respect to the transcription start site at +1), with polyamide binding sites at various distances upstream (U) or downstream (D) from the TATA box (boxed). Polyamide-binding models are shown.

FIG. 1.

Polyamide structures and binding sites. (A) Structures for polyamides ImPy-β-ImPy-γ-ImPy-β-ImPy-β-Dp (1), ImIm-β-ImIm-γ-PyPy-β-PyPy-β-Dp (2), and ImPy-β-PyIm-daba-PyIm-β-ImPy-β-Dp (3). Base sequence specificity depends on side-by-side pairing of Py and Im amino acids in the minor groove of DNA (6). Im-Py targets G·C base pairs and Py-Im targets C·G base pairs. Py-Py is degenerate and targets both A·T and T·A base pairs. The β-alanine-β-alanine pair also recognizes both A·T and T·A base pairs (42). Black and white circles, Im and Py rings, respectively; curved line, hairpin junction, which is formed with γ-aminobutyric acid or daba; diamonds, β-alanine; parenthesis with plus sign, Dp. The positive charge on the daba turn amino acid (3) is not shown. Me, methyl. (B) DNA sequences of the HIV-1 promoter constructs (from nucleotide positions −55 to +1, with respect to the transcription start site at +1), with polyamide binding sites at various distances upstream (U) or downstream (D) from the TATA box (boxed). Polyamide-binding models are shown.

FIG. 2.

DNase I footprint analysis of polyamide 3 binding. (A) DNase I footprint of two upstream constructs, U+1 (lanes 1 to 10) and U−10 (lanes 11 to 20). Lanes 1 and 11, DNA alone; lanes 2 to 10 and 12 to 20, polyamide 3 at 1, 3, 5, 10, 15, 20, 30, 40, and 50 nM, respectively. The locations of the polyamide binding sites, TATA box, transcription start site (denoted +1), and direction of transcription (arrow) are indicated. The match site at −113 to −119 is also indicated. ³²P, end label on the DNA probe. (B) Graphical representation of polyamide 3 titration plotted as the fraction of DNA bound (normalized to the fraction bound in the absence of polyamide) versus polyamide concentration.

FIG. 3.

Affinity cleavage with polyamide 3-EDTA·Fe(II). (A) Each of the indicated radiolabeled DNAs (at 40 pM) was incubated with polyamide at a final concentration of 5 nM (lanes +) or without polyamide (lanes −), and affinity cleavage reactions were performed as described in Materials and Methods. G+A sequencing reactions are also shown. The location of the TATA box (vertical bar) and nucleotide positions relative to T+1 of the TATA box are indicated. Arrow, location of the transcription start site and direction of transcription; ∗, match site at nucleotide positions −113 to −119, relative to the transcription start site, in each of the constructs. (B) DNA sequences of the HIV-1 promoter constructs (from nucleotide positions −44 to −6, with respect to the transcription start site). Polyamide-binding sites are in boldface, and the TATA sequence is boxed. Vertical lines, results for affinity cleavage with polyamide 3-EDTA·Fe(II), with the height and width of each line proportional to cleavage intensity at a given site.

FIG. 3.

Affinity cleavage with polyamide 3-EDTA·Fe(II). (A) Each of the indicated radiolabeled DNAs (at 40 pM) was incubated with polyamide at a final concentration of 5 nM (lanes +) or without polyamide (lanes −), and affinity cleavage reactions were performed as described in Materials and Methods. G+A sequencing reactions are also shown. The location of the TATA box (vertical bar) and nucleotide positions relative to T+1 of the TATA box are indicated. Arrow, location of the transcription start site and direction of transcription; ∗, match site at nucleotide positions −113 to −119, relative to the transcription start site, in each of the constructs. (B) DNA sequences of the HIV-1 promoter constructs (from nucleotide positions −44 to −6, with respect to the transcription start site). Polyamide-binding sites are in boldface, and the TATA sequence is boxed. Vertical lines, results for affinity cleavage with polyamide 3-EDTA·Fe(II), with the height and width of each line proportional to cleavage intensity at a given site.

FIG. 4.

Inhibition of TBP binding to the TATA box by polyamide 3. (A) DNase I footprint of the radiolabeled D+6 DNA in the presence of DNA alone (lane 2); 40 nM polyamide 3 (lane 3); TBP (lane 4); and TBP and polyamide 3 at 3, 5, 10, 15, 20, 30, and 40 nM (lanes 5 to 11, respectively). Lane 1, G+A sequencing ladder. (B) Co-occupancy of TBP and polyamide 3. DNase I footprint of D+10 in the presence of DNA alone (lane 2); 40 nM polyamide 3 (lane 3); TBP (lane 4); and TBP and polyamide 3 at 3, 5, 10, 20, 30, and 40 nM (lanes 5 to 10, respectively). Lane 1, G+A sequencing ladder.

FIG. 5.

The effects of distance on inhibition of TBP binding to the TATA box by polyamide 3. Shown is a graphical representation of footprint analysis of polyamide inhibition plotted as fraction of DNA bound (normalized to the fraction of DNA bound in the absence of polyamide) versus polyamide concentration. (A) Upstream constructs. (B) Downstream constructs. (C) Polyamide-binding affinity in the presence and absence of TBP for the D+6 construct.

FIG. 6.

Inhibition of the ternary TBP-TFIIA-DNA complex by polyamide 3. (A) DNase I footprint of U−2 DNA in the presence of DNA alone (lane 2); TBP (lane 3); TBP plus TFIIA (lane 4); TBP, TFIIA, and polyamide 3 at 3, 10, 15, 20, 30, and 50 nM (lanes 5 to 10, respectively). All reaction components were incubated simultaneously for 30 min prior to digestion with DNase. Lane 1, G+A sequencing ladder; lane 11, 50 nM polyomide 3, no TBP or TFIIA. The location of the TATA box and polyamide-binding site are indicated. (B) Graphical representation of inhibition plotted as the fraction of DNA bound (normalized to the fraction of TBP bound in the absence of polyamide) versus polyamide concentration.

FIG. 7.

Inhibition of basal transcription. (A) Schematic representation of wild-type and mutant construct transcripts. The polyamide binding sites, TATA box, and approximate transcription start sites are indicated as for Fig. 1. (B) Runoff transcription of wild-type and D+6 constructs monitored with a HeLa nuclear extract (see Materials and Methods). Lanes 1 to 3, wild-type DNA; lanes 4 to 9, D+6 DNA. The DNA was incubated with no polyamide (lane 1); 500 nM polyamide 1 (lane 2); 500 nM mismatch polyamide 2 (lane 3); no polyamide (lane 4); polyamide 3 at 50, 100, 300, and 500 nM (lanes 5 to 8, respectively); and 500 nM mismatch polyamide 2 (lane 9). (C) Graphical representation of inhibition plotted as percent transcription (normalized to the no-polyamide control reaction) versus polyamide concentration for D+6. (D) Relative transcription signals for each of the indicated DNA constructs (Fig. 1B) are plotted versus polyamide 3 concentration. Transcription levels for each template were normalized to the level observed in the absence of polyamide.

FIG. 8.

Polyamide 3 inhibition of the TFIID-TFIIA-DNA complex analyzed by EMSA. (A) Radiolabeled D+15 DNA was incubated with either no polyamide (lanes −) or with 25, 50, and 200 nM polyamide 3 or 200 nM polyamide 1 for 15 min prior to the addition of TFIID plus TFIIA, as indicated. After a subsequent 20-min incubation, the samples were subjected to electrophoresis. Only the region of the gel containing the TFIID-TFIIA-DNA (DA-DNA) complex is shown. (B) Radiolabeled U−15 DNA was incubated with either no polyamide or with 50, 100, and 200 nM polyamide 3 or 200 nM polyamide 2 for 15 min prior to the addition of TFIID plus TFIIA, as for panel A. (C) Phosphorimage quantitation of the extent of DA-DNA complex formation with increasing concentrations of polyamide 3. Data are normalized to the phosphorimage units in the DA-DNA complex in the absence of polyamide.