Use of Site-Specific Protein–DNA Photocrosslinking to Analyze the Molecular Organization of the RNA Polymerase II Initiation Complex

1. Introduction

Site-specific protein–DNA photocrosslinking has proved to be the method of choice for analysis of the formation of nucleoprotein complexes such as those involved in transcription by mammalian RNA polymerase II (RNA Pol II). The method has two principal advantages. First, it yields structural information on large, multisubunit complexes that in general cannot be analyzed using standard high-resolution techniques such as X-ray crystallography or nuclear magnetic resonance (NMR). For example, site-specific protein–DNA photocrosslinking, in conjunction with complementary methods such as protein-affinity chromatography and electron microscopy, has produced information on both the molecular organization and the composition of the RNA Pol II pre-initiation complex on promoter DNA (1–4). This complex contains RNA Pol II and the general transcription factors TBP, TFIIA, TFIIB, TFIIE, TFIIF (RAP74 and RAP30), and TFIIH, and is composed of more than 25 polypeptides ranging in M_r from 10 to 220 kDa (5). Neither X-ray crystallography nor NMR, which can only resolve the structure of complexes containing short protein fragments bound to small pieces of promoter DNA, could provide any detailed structural information on this complex. Second, the method has sufficient technical flexibility so as to allow the rapid analysis of complexes assembled under various conditions. Over the past few years, we have analyzed a large collection of complexes assembled in the presence of various combinations of the general transcription factors (wild-type or different deletion mutants) and RNA Pol II (1–4). These experiments have enabled us to draw conclusions on the dynamics of RNA Pol II pre-initiation complex assembly and has led to the notion that isomerization of the RNA Pol II pre-initiation complex proceeds through wrapping of the promoter DNA around the enzyme (4).

Site-specific protein–DNA photocrosslinking is a method composed of two successive steps. First, a number of photoprobes that place one (or a few) photoreactive nucleotide(s) into juxtaposition with one (or a few) radiolabeled nucleotide(s) at various specific positions along the promoter DNA are prepared. Second, transcription complexes are assembled onto the various photoprobes, irradiated with ultraviolet (UV) light so as to induce protein–DNA crosslinking, and the processed in order to identify the crosslinked polypeptides. Because the crosslinking of protein to DNA is site-specific, the use of a series of photoprobes that place the photonucleotide derivative at various positions along the promoter DNA provides information on the relative position of the various factors within the complex.

2. Materials

1. Buffer A (10X): 300 mM Tris-HCl, pH 8.0, 500 mM KCl, and 70 mM MgCl₂, freshly prepared.

2. Bovine serum albumin (BSA) solution: Prepare a 25-mg/mL solution of BSA in deionized distilled water. Store in aliquots at −20°C.

3. Dilute with water to 5 mg/mL prior to use.

4. dNTP mix: 20 mM each of dATP, dCTP, dGTP, and dTTP in buffer A (1X), freshly prepared.

5. ND buffer: 20 mM HEPES, pH 7.9, 100 mM KCl, 20% glycerol, 0.2 mM EDTA, 0.2 mM EGTA, and 10 mM of β-mercaptoethanol. Store in aliquots at −20°C.

6. TBE buffer (10X): Prepare 1 L by mixing 108 g Tris base, 55 g boric acid, and 40 mL EDTA (0.5 M, pH 8.0).

7. Gel loading solution (10X): For polyacrylamide native gels, use a solution containing 0.25% bromophenol blue, 0.25% xylene cyanol, and 25% Ficoll (type 400) in deionized distilled water.

8. MBS (5X): 40 mM MgCl₂, 100 mM HEPES, pH 7.9, 100 μg/mL BSA, and 5 mM ATP.

9. Store in aliquots at −20°C.

10. Complex mix: 50 μL MBS (5X) and 2 μL (NH₄)₂SO₄ (2 M), freshly prepared.

11. Poly(dI.dC–dI.dC) stock: Prepare a 25-mg/mL solution of poly(dI.dC–dI.dC) in deionized distilled water. Store in aliquots at −20°C.

12. DNase mix: A solution containing 200 units/mL DNase I and 32 mM CaCl₂, freshly prepared.

13. Acid mix: Mix equal volumes of 5% acetic acid and 30 mM ZnCl₂, freshly prepared.

14. S1 mix: 30,000 U/mL S1 nuclease in deionized distilled water, freshly prepared.

15. Dilution mix: 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 10 mM EGTA, 500 mM NaCl, 5 mM NaF, 1 mM phenymethylsulfonyl fluoride (PMSF), and 1% (v/v) Triton X-100, freshly prepared.

16. Protein A mix: 100 mg/mL Protein A in the dilution mix.

17. Wash 1 solution: 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 1 mg/mL BSA, 0.5% NaN₃, and 0.5% Na-deoxychlorate, freshly prepared.

18. Wash 2 solution: 10 mM Tris-HCl, pH 8.0, and 1.5 M NaCl.

19. Wash 3 solution: 10 mM Tris-HCl, pH 8.0, and 150 mM NaCl.

3. Methods

3.1. Day 1: Synthesis and Purification of the Photoprobes

The first step of the procedure is the synthesis of the photoprobes. This part is illustrated in Fig. 1 which shows, as an example, a scheme for the synthesis of a photoprobe designed to place the photoreactive nucleotide at position −2 relative to the transcriptional initiation site of the adenovirus major late promoter. The site-specific incorporation of the photoreactive nucleotide (see ^{Note 1}) and the radiolabeled nucleotide is directed through the annealing of a primer, referred to as the specific primer, with a single-stranded DNA template containing the promoter DNA. The promoter is flanked by two restriction sites (in this example, DraI and SacI). A second primer, referred to as the upstream primer, is annealed a few base pairs upstream of the DraI site. After annealing, the photoreactive and radiolabeled nucleotides are incorporated by primer extension using T₄ DNA polymerase with limiting amounts of dNTPs (see ^{Note 2}). After the labeling step, the extension reaction is completed by the addition of an excess of cold dNTPs (see ^{Note 3}). The photoprobe is generated by digestion with the restriction enzymes and gel purified (see Fig. 2 for an example of a gel on which the products of a photoprobe synthesis reaction have been separated).

Fig. 1.

Synthesis and purification of the photoprobes. Schematic representation of the synthesis of a photoprobe that places a photoreactive nucleotide at position −2 and three radiolabeled nucleotides at positions −4, −3, and −1 of the adenovirus major late promoter. The sequence of the adenovirus major late promoter flanked by plasmid DNA is shown. The primers used to direct the incorporation of N₃R–dUMP (U) and radiolabeled nucleotides (*) are shown in boxes. The TATA element is in bold type and the transcriptional initiation site is indicated by an arrow. DraI and SacI restriction sites used for the excision of the photoprobe are indicated. In the first step, the primers are annealed to single-stranded DNA containing the promoter sequence (Annealing). In the second step, the photoreactive and the radiolabeled nucleotides are incorporated using T₄ DNA polymerase (Labeling). In the third step, the extension reaction is completed by a chase with excess of cold dNTPs (Chase). In the fourth step, the site-specifically labeled promoter fragment is excised using restriction enzymes and gel purified (Digestion and purification).

Fig. 2.

Example of the autoradiogram of a gel after electrophoresis of the products of a synthesis reaction. The positions of the photoprobe, the free nucleotides, and the plasmid fragment are shown. The photoprobe is then purified from the gel.

• 1Mix 700 ng of single-stranded (ss) DNA (approx 0.5 pmol) with 40 ng (approx 5 pmol) of both the specific and upstream primers. Add 1 μL of Buffer A (10X) and complete to 10 μL with deionized distilled water.
• 2Mix well and incubate for 3 min at 90°C.
• 3Incubate for 30 min at room temperature.
• 4From this point on, all manipulations must be carried out under reduced light conditions (see ^{Note 4}). Add 1 μL BSA (5 mg/mL), 1 μL N₃R–dUMP (see ^{Note 1}), 25 μCi of the appropriate [α-³²P]–dNTP ([α-³²P]–dCTP for the example shown in Fig. 1), 5–10 U T4 DNA polymerase, and 1 μL buffer A (10X). Complete to a final volume of 20 μL with deionized distilled water.
• 5Incubate for 30 min at room temperature.
• 6Add 5 μL of dNTP mix.
• 7Incubate for 5 min at room temperature.
• 8Incubate for 20 min at 37°C.
• 9Add 10–20 U of each restriction enzyme (SacI and DraI in the example shown in Fig. 1).
• 10Incubate for 90 min at the temperature recommended by the supplier of restriction enzymes (37°C for SacI and DraI).
• 11Add 3.5 μL of gel loading solution (10X).
• 12Load on a native 8% polyacrylamide gel in TBE buffer (1X).
• 13Run at 150 V for about 1 h in TBE buffer (1X).
• 14Remove the glass plates containing the gel from the gel box.
• 15Separate the glass plates and leave the gel on one of them.
• 16Wrap the gel/glass plate in plastic wrap.
• 17Wrap the entire package in aluminum foil.
• 18Move to a dark room (see ^{Note 5}).
• 19Place a Kodak X-OMAT AR film on a clean bench.
• 20Remove the foil and place the gel on the film with the glass plate facing up (e.g., gel side down).
• 21Expose 5 min.
• 22During the exposition time, mark the film using a sharp tool by tracing the contour of the glass plate (this will be helpful later for the localization of the photoprobe in the gel).
• 23Remove the gel and rewrap it with the foil.
• 24Develop the film (see ^{Note 6}).
• 25Using a scalpel, cut the film so that the square piece containing the band corresponding to the photoprobe is removed. This operation leaves the film with a window at the position of the photoprobe.
• 26Superimpose the film on the gel by taking advantage of the marks made in step 22, and mark the square corresponding to the photoprobe on the saran wrap using a pen.
• 27Cut out the gel slice containing the photoprobe using a clean scalpel.
• 28Cut the gel slice in small pieces (six to eight fragments).
• 29Place the gel fragments in an Eppendorf tube and add water in order to completely submerge the gel (usually 100–150 μL of water).
• 30Incubate overnight at room temperature.
• 31Collect the liquid containing the probe.
• 32Purify the probe on a Micro-Spin S-200 HR column (Pharmacia Biotech) to remove any salts and other putative contaminants.
• 33Count 1 μL of the photoprobe solution by liquid scintillation, and dilute the probe to 1250 cpm/μL with deionized distilled water.
• 34The probe is ready for use and can be stored in the dark at 4°C for 1–2 wk (see ^{Note 7}).

3.2. Day 2: Protein–DNA Photocrosslinking

The gel-purified photoprobe is used for pre-initiation complex assembly with the purified transcription factors (TBP, TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH) and RNA Pol II. Reactions are irradiated with ultraviolet (UV) light to induce protein–DNA crosslinking and treated with DNase I and S1 nuclease in order to liberate polypeptides that are covalently attached to a very short piece of DNA carrying one to four radiolabeled nucleotides. After sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) separation of the photocrosslinked polypeptides, the gel is dried and exposed to X-ray film. Examination of the ensuing autoradiogram permits identification of the protein(s) that interact with a particular site. For the RNA Pol II initiation complex, the specificity of the photocrosslinking signals can be assessed by comparing reactions performed with photoprobes containing either a wild-type or a mutated TATA element and/or by comparing reactions performed in either the presence or the absence of TBP (see ^{Note 8}). The photocrosslinked polypeptides can be identified according to their M_r (Fig. 3A,B) and to their immunoreactivity with specific antibodies (Fig. 3C). In the latter case, crosslinking reactions are submitted to immunoprecipitation with antibodies directed against specific factors prior to SDS-PAGE (see ^{Note 9}).

Fig. 3.

Typical SDS-PAGE gels of photocrosslinked proteins. (A) Crosslinking reactions assembled either in the absence of TBP or using a probe with a mutated TATA box give identical results. Crosslinking reactions were performed with TFIIB, RAP30, RAP74, TFIIE34, TFIIE56, and RNA Pol II in the presence (+) or in the absence (−) of TBP using either a wild-type (TATAAA) or a mutated (TAGAGA) TATA element. In the example shown here (the photonucleotide derivative is placed at position −15 of the adenovirus major late promoter), the crosslinking of both the second largest subunit of RNA Pol II (RPB2) and RAP74 are considered to be specific (dark arrowhead). The crosslinking of RPB1 is considered as nonspecific (open arrowhead) because it is not affected by the omission of TBP or the use of a probe with a mutation in the TATA box. The positions of the molecular weight markers (MW) are indicated. (B) The use of truncated polypeptides as a tool to identify the photo-crosslinked polypeptides. Crosslinking reactions were performed using TFIIB, RAP30, TFIIE34, TFIIE56, and RNA Pol II in the presence (+) or in the absence (−) of TBP, using full-length (1–517) or truncated (1–409) RAP74. The different mobilities of RAP74 fragments are diagnostic for RAP74 contact at this promoter position. (C) Immuno-precipitation of photocrosslinked polypeptides. Crosslinking reactions were performed using TFIIB, RAP30, RAP74, TFIIE34, TFIIE56, and RNA Pol II in the presence (+) or in the absence (−) of TBP. The photocrosslinked polypeptides were either processed normally (first two lanes) or immunoprecipitated using an antibody directed against RAP74 or a control antibody.

• 35Mix the proteins (50–200 ng each) and complete the volume to 16 μL with ND buffer (see ^{Note 10}). Add 5.2 μL of the complex mix, 1 μL of diluted poly(dI.dC–dI.dC) (see ^{Note 11}) and 5000 cpm of the photoprobe (4 μL of 1250 cpm/μL). The final volume is 26.2 μL.
• 36Mix well and incubate for 30 min at 30°C.
• 37Open the lids of the tubes and irradiate 10 min with UV light (see ^{Notes 12} and ¹³).
• 38Add 5 μL of DNase mix.
• 39Incubate for 20 min at 37°C.
• 40Add 1.5 μL of 10% SDS.
• 41Incubate for 3 min at 90°C.
• 42Spin for 10 s in a microfuge (16,000g)
• 43Add 2 μL of Acid mix.
• 44Add 1 μL of S1 mix.
• 45Incubate for 20 min at 37°C.
• 46For regular reactions, go directly to step 62.
• 47For immunoprecipitation reactions, add 315 μL of the dilution mix.
• 48Add 5 μL of antibody (see ^{Note 14}).
• 49Incubate for 60 min at 4°C.
• 50Add 80 μL of Protein A mix.
• 51Agitate 60 min at 4°C using a rocker.
• 52Spin down the Protein A beads (1 min in a microfuge at 16,000g).
• 53Remove the supernatant by pipetting.
• 54Wash with 400 μL of wash 1 solution.
• 55Repeat steps 52–54 twice.
• 56Wash with 400 μL of wash 2 solution.
• 57Spin down the Protein A beads (1 min in a microfuge at 16,000g).
• 58Remove the supernatant by pipetting.
• 59Wash with 400 μL of wash 3 solution.
• 60Spin down the Protein A beads (1 min in a microfuge at 16,000g).
• 61Remove the supernatant by pipetting.
• 62Add SDS gel loading solution and boil 5 min (see ^{Note 15}).
• 63Resolve the photocrosslinked polypeptides by SDS-PAGE (run at 30 mA in the stacking gel and 50 mA in the separating gel) (see ^{Note 15}).
• 64Transfer the gel to Whatman paper and dry.
• 65Expose the dried gel to X-ray film using an intensifying screen (see ^{Note 16}).