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Analysis of Yeast RNAP I Transcription of Nucleosomal Templates In Vitro

Part of the Methods in Molecular Biology book series (MIMB,volume 2533)

Abstract

Nuclear eukaryotic RNA polymerases (RNAPs) transcribe a chromatin template in vivo. Since the basic unit of chromatin, the nucleosome, renders the DNA largely inaccessible, RNAPs have to overcome the nucleosomal barrier for efficient RNA synthesis. Gaining mechanistical insights in the transcription of chromatin templates will be essential to understand the complex process of eukaryotic gene expression. In this article we describe the use of defined in vitro transcription systems for comparative analysis of highly purified RNAPs I–III from S. cerevisiae (hereafter called yeast) transcribing in vitro reconstituted nucleosomal templates. We also provide a protocol to study promoter-dependent RNAP I transcription of purified native 35S ribosomal RNA (rRNA) gene chromatin.

Key words

  • Transcription
  • Chromatin
  • Nucleosomes
  • Histones
  • RNA polymerase I
  • RNA polymerase II
  • RNA polymerase III
  • Ribosomal RNA gene chromatin
  • Rrn3
  • Core factor
  • Net1
  • In vitro assays
  • Saccharomyces cerevisiae
  • Yeast

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1 Introduction

In all eukaryotes there are at least three different nuclear multisubunit RNA Ps (RNA polymerases) termed I–III (reviewed in [1], see also short reviews from Merkl et al., and Pilsl et al. in this issue). The enzymes share common subunits and a similar core structure but clearly differ in their composition and function (reviewed in [1], see also short reviews from Merkl et al., and Pilsl et al. in this issue). The template transcribed by RNAPs I–III in vivo is chromatin . In chromatin , approximately 146 bp of DNA are wrapped around an octameric core of histone proteins forming a basic repeating unit called the nucleosome (reviewed in [2,3,4]). This leads to compaction and impairs access to the genetic information. Therefore, chromatin structure must be transiently altered allowing transcription at defined genomic loci. Accordingly, characteristic chromatin transitions have been observed in vivo, when genes switch from an transcriptionally inactive to an actively transcribed state (reviewed in [5, 6]). There is evidence that RNAPs I–III deal differently with the chromatin template ([7], see also short reviews from Merkl et al. and Schächner et al. in this issue).

Defined in vitro systems including purified components contributed substantially to our current understanding of mechanisms of eukaryotic transcription (reviewed in [8]). The possibility to use in vitro reconstituted nucleosomal templates provided a minimal model system for transcription in the context of chromatin (reviewed in [9]). Here, we describe a protocol for comparative analyses of yeast RNAPs I–III in promoter-independent transcription of in vitro reconstituted chromatin templates. To this end, we developed a purification procedure for affinity purification of the three enzymes under identical conditions [10, 11]. The purified enzymes were devoid of cross-contamination with other RNAPs or transcription factors . For promoter-independent transcription , tailed DNA templates containing a 3′ overhang can be employed (Fig. 1a) [12]. From 3′ overhangs all RNAPs can initiate transcription without additional transcription factors . These templates in combination with the strong 601 nucleosome positioning sequence (601 templates) [13] were used for nucleosome assembly at defined positions (Fig. 1b). To determine the transcription efficiency through an assembled nucleosome , radioactively labeled RNA synthesized by the different RNAPs from the nucleosomal 601 templates are detected and quantified (Fig. 1c, d). Each transcription reaction contains another nucleosome-free reference template of different size, which serves as an internal control for the transcriptional activity of the respective RNAP. Additionally, transcription reactions are performed containing the naked 601 templates and the reference template (Fig. 1c). The transcription efficiency through a nucleosome is then calculated by dividing the quotient of transcripts from nucleosomal 601 template to reference template by the quotient of transcripts from naked 601 template to reference template. These analyses revealed marked differences between transcription of nucleosomal templates by RNAPs I–III ((Fig. 1d), and [7]). Whereas RNAPs I and III could readily transcribe through an in vitro assembled nucleosome RNAP II transcription was strongly impaired.

Fig. 1
figure 1

Promoter-independent transcription of in vitro assembled nucleosomal templates by RNAPs I–III. (a) Schematic representation of the template for nucleosome assembly containing a 601 nucleosome positioning sequence (601 template). The reference template lacks the nucleosome positioning sequence but is otherwise identical. Restriction sites used to release the templates from plasmids K1253 and K1573 are indicated. (b) Electrophoretic mobility shift assay after nucleosome assembly. Purified core histones H2A, H2B, H3, and H4 from chicken erythrocytes were assembled on the 601 template shown in (a) by salt gradient dialysis. Assembly reactions with different histone to DNA ratios (Table 7) were analyzed in a native polyacrylamide gel as described in Subheading 3.2. The position of the nucleosome-free template and the K1253 vector backbone and the chromatin template after assembly are indicated on the left. Sizes of selected DNA fragments in DNA markers (M) are indicated on the right. (c, d) Purified RNAPs I–III transcribe through a nucleosome on tailed templates with different efficiencies. (c) Transcription reactions were performed in the presence of a reference template and the 601 template before (−) or after (+) nucleosome assembly. The cartoon on the left indicates the length of the transcripts derived from the different templates. In vitro transcription assays were performed using 7.5 nM of purified RNAPs, identical buffer conditions and 10 nM of each of the different templates. Radiolabeled transcripts were separated on a denaturing polyacrylamide gel and visualized as described in Subheading 3.5.2. Sizes of selected RNA fragments in an RNA marker (M) are indicated on the left. (d) Quantification of the experiments shown in (c) was performed as described in Subheading 3.5.3

Nucleosomal templates obtained after salt dialysis have the advantage that they are fully defined. However, it is unclear in how far they reflect endogenous chromatin , the native template of nuclear transcription . Thus, we established a technique allowing the excision of a genomic region of interest in its native chromatin context by site-specific recombination in specifically engineered yeast strains (Fig. 2a) [17,18,19]. To assist affinity purification of the released chromatin domain, the genomic region of interest contains a cluster of DNA binding sites for the bacterial LexA protein . In the yeast strains used for recombination, recombinant LexA C-terminally fused to a tandem affinity purification (TAP ) tag is constitutively expressed. The protein binds to its binding sites within the chromatin domain and serves as bait for subsequent affinity purification of the genomic target region (Fig. 2b). The isolated native chromatin domains are suitable substrates for compositional analysis by mass spectrometry , structural analysis by electron microscopy or biophysical molecular tweezer analysis [20,21,22,23]. Furthermore, they can be used in functional assays to study transcription factor binding, chromatin remodeling, or transcription on native templates in vitro [18, 24, 25]. Here, we show that purified native 35S rRNA gene chromatin can be used as template for promoter-dependent RNAP I transcription . To this end, the chromatin domain bound to the affinity matrix is linearized by restriction enzyme digest to produce transcripts of a defined length (Fig. 2c). Promoter-dependent transcription of the purified chromatin domain requires at least purified RNAP I , as well as two components of the RNAP I transcription machinery, the three-subunit core factor (CF) and the initiation factor Rrn3 (Fig. 2d). We find that the Net1 protein, which has previously been characterized as an activator of RNAP I transcription in vitro and in vivo [15, 26], leads to a robust stimulation of RNAP I dependent native chromatin transcription (Fig. 2d).

Fig. 2
figure 2

Promoter-dependent transcription of purified native rRNA gene chromatin by RNAP I. (a) Schematic representation of purification and restriction enzyme digest of yeast native 35S rRNA gene chromatin . (b) DNA analysis of samples withdrawn during the purification procedure (shown in a, and described in Subheadings 3.3.1 and 3.3.2). DNA of the indicated fraction of individual samples depicted on the top was isolated as described in Subheading 3.3.4, linearized with SacII and separated by electrophoresis in a 1% agarose gel. An image of the agarose gel stained with SYBR safe is shown on the top. The same gel was subjected to the Southern blot procedure with a radioactively labeled probe described in Subheading 2.3. The autoradiogram of the blot is shown at the bottom. The positions and respective length of DNA marker fragments (NEB 1 kb ladder, not shown) are depicted on the left of the gel picture. The position of the rDNA fragment derived from the 35S rRNA gene chromatin domain in the gel and on the blot is depicted on the right. (c) Quantitative Southern blot analysis of the “beads post EcoRV digest” sample (BpD) together with a titration from 0.1 to 1 fmol of plasmid K375. The isolated DNA was digested with PflmI prior to electrophoresis in a 1.5% agarose gel which was subjected to the Southern blot procedure. The positions and respective length of DNA marker fragments (NEB 1 kb ladder, not shown) are depicted on the left of the autoradiogram. The positions of PflmI fragments derived from the EcoRV digested or undigested 35S rRNA gene chromatin domain, as well as from the PflmI/EcoRV digested plasmid K375 are depicted on the right of the autoradiogram. (d) Promoter-dependent in vitro transcription of native rRNA gene chromatin . Promoter-dependent in in vitro transcription was performed as described in [14] using either 10 nM of EcoRV (E) or PvuII (P) linearized plasmid K375 or 10 nM of EcoRV linearized rRNA gene chromatin as template. Note that only 20% of the chromatin template were properly digested by EcoRV and produce transcripts with a defined length. Individual transcription reactions were performed in the presence or absence of RNAP I (5 nM), CF (20 nM), Rrn3 (70 nM), and the RNAP I transcription activator Net1 (20 nM) [15, 16], as indicated on top. Radiolabeled transcripts were separated on a denaturing polyacrylamide gel and visualized as described in Subheading 3.5.2. The positions and respective lengths of RNAs synthesized from PvuII or EcoRV digested plasmid K375 or chromatin templates are indicated on the left and the right of the autoradiogram

In summary, combining analyses involving strictly defined in vitro assembled nucleosomal templates and ex vivo purified native chromatin will likely help to gain further insights in mechanisms of nuclear transcription by eukaryotic RNAPs.

2 Materials

2.1 Preparation of Tailed Templates

Details about construction of plasmids K1253 (pUC19 tail g—601) and K1573 (pUC19 tail g—w/o BS) can be found elsewhere [10, 11]. Plasmids and related sequence information are available upon request.

  1. 1.

    Plasmid K1253 for nucleosome reconstitution, and reference plasmid K1573.

  2. 2.

    Nb. BsmI, KasI, PvuII, and respective buffers (NEB).

  3. 3.

    RNase-free water.

  4. 4.

    Competitor oligo o2207, with the same sequence

    (5′-CGAGTAAGTATAGGGTAAGGTGAT-3′) as the 24 nt overhang generated by Nb.BsmI/KasI digest of plasmid K1253 or K1573.

  5. 5.

    Thermocycler.

  6. 6.

    NanoDrop spectrophotometer (Thermo Fisher Scientific).

2.2 In Vitro Nucleosome Assembly

  1. 1.

    Purified core histones H2A, H2B, H3 and H4 from chicken erythrocytes (prepared as described in [27]).

  2. 2.

    Bovine serum albumin (10 mg/mL), RNase free.

  3. 3.

    DNA template (see Subheading 3.1).

  4. 4.

    Siliconized micro tubes (Eppendorf).

  5. 5.

    Buffers see Table 1.

  6. 6.

    Polyacrylamide gel for electrophoretic mobility shift assay (EMSA) (Table 2).

  7. 7.

    6× EMSA buffer (60% glycerol, Orange G).

  8. 8.

    FLA3000 (Fuji) Imager or equivalent imaging system for Ethidium bromide staining.

  9. 9.

    Gel dryer (Drystar).

  10. 10.

    Whatman filter paper (Macherey-Nagel, MN 827 B).

Table 1 Buffers for chromatin assembly
Table 2 Composition of native polyacrylamide gel for EMSA

2.3 Purification and Restriction Enzyme Digest of Native Yeast Chromatin

Materials for strain construction, cell growth and harvesting (preparation of yeast cell “spaghetti”), as well as for coupling of rabbit immunoglobulin G (IgG) to epoxy-activated magnetic beads , and protein analysis have been described elsewhere [19, 20]. Strain y2381 used for the purification of a 35S rRNA gene chromatin domain and plasmid K375 which contains an entire rDNA repeat were used in the experiments shown in Fig. 2 and have been described earlier [20, 28]. The template to obtain the radioactively labeled probe in the Southern blots shown in Fig. 2b, c was obtained by PCR from plasmid K358 (pM49.2) [17] with primers o597 (5′-GAAACAGCTATGACCATG-3′) and o598 (5′-GCCTGACTGCAGAAC GTACTACTGTACATATAAC-3′). Strains, plasmids as well as related sequence information are available upon request.

2.3.1 Preparation of Cellular Lysates

  1. 1.

    Coffee grinder (Tefal Prep’Line).

  2. 2.

    PARAFILM® M.

  3. 3.

    Proteinase inhibitors (100× PIs): benzamidine (33 mg/mL), PMSF (17 mg/mL) in ethanol p.a., stored at −20 °C.

  4. 4.

    Dry ice pellets (150 g per purification).

  5. 5.

    Frozen yeast cell “spaghetti” (3 g per purification).

  6. 6.

    Magnetic bead buffer (MB100): 20 mM Tris-HCl pH 8.0, 100 mM KCl, 5 mM MgAc, 0.5% Triton X-100 (w/v), 0.1% Tween 20 (w/v). Buffer MB (without additives) can be stored at room temperature and should be cooled to 4 °C prior to use. 2-Mercaptoethanol (final concentration 1.5 mM) and proteinase inhibitors (final concentration 1× PIs) should only be added prior to use.

  7. 7.

    Bio-Rad Protein Assay Dye Reagent Concentrate.

2.3.2 Affinity Chromatography of Native Chromatin Domains

  1. 1.

    IgG coupled to magnetic beads , prepared as previously described [19].

  2. 2.

    BcMag Separator (Bioclone Inc., MS-04).

  3. 3.

    Rotating wheel or alternative rotation device.

  4. 4.

    Magnetic bead Buffer (MB100), as described in Subheading 3.3.1.

2.3.3 Restriction Enzyme Digest of Purified Native Chromatin Domains

  1. 1.

    Restriction enzymes and respective buffer (NEB).

  2. 2.

    Thermomixer® Dry Block Heating Shaker (Eppendorf).

2.3.4 DNA Analysis

  1. 1.

    Buffer IRN: 50 mM Tris-HCl, pH 8.0, 20 mM EDTA, 0.5 M NaCl.

  2. 2.

    Buffer TE: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA.

  3. 3.

    RNase A (20 mg/mL) (Invitrogen).

  4. 4.

    Proteinase K (20 mg/mL) (Sigma), dissolved in 50 mM Tris-HCl pH 8.0, 1 mM CaCl2 and stored in aliquots at −20 °C.

  5. 5.

    Yeast tRNA (10 mg/mL) (Sigma), dissolved in nuclease-free water.

  6. 6.

    Roti® phenol–chloroform–isoamyl alcohol (25:24:1) (Roth), stored at 4 °C in the dark.

  7. 7.

    Ethanol p.a., stored at room temperature.

  8. 8.

    Restriction enzymes and respective buffer (NEB).

2.4 Purification of RNAPs I, II, and III from S. cerevisiae

  1. 1.

    Vibrax-VXR (IKA), rotary shaker.

  2. 2.

    IgG coupled to magnetic beads , and BcMag Separator, rotating wheel as described in Subheading 3.3.2.

  3. 3.

    Thermomixer® Dry Block Heating Shaker (Eppendorf).

  4. 4.

    Glass beads (ø 0.75–1 mm) (Roth).

  5. 5.

    YPD medium: 2% (w/v) Bacto™ Peptone, 1% (w/v) Bacto™ Yeast Extract, 2% (w/v) Glucose.

  6. 6.

    Proteinase inhibitors (100×), as described in Subheading 3.3.1.

  7. 7.

    Purified recombinant 6× His-tagged Tobacco Etch Virus (TEV) protease produced in E. coli .

  8. 8.

    Yeast strains (Table 3).

  9. 9.

    Buffers (Table 4).

Table 3 Yeast strains used for RNA polymerase purification
Table 4 Buffers for RNA polymerase purification

2.5 In Vitro Transcription of Chromatin Templates

  1. 1.

    Buffers for in vitro transcription (Table 5).

  2. 2.

    Buffers for denaturing polyacrylamide gel electrophoresis (Table 6).

  3. 3.

    GelSave (Applichem).

Table 5 Buffers for in vitro transcription experiments
Table 6 Composition of denaturing polyacrylamide gel for RNA electrophoresis

3 Methods

3.1 Preparation of Tailed Templates

  1. 1.

    Fragments for tailed template preparation are released from 10 μg plasmid K1253 or the reference plasmid K1573 using restriction enzymes PvuII and KasI for 1.5 h at 37 °C in a total volume of 100 μL, followed by heat inactivation of these enzymes at the appropriate temperature as indicated by the manufacturer.

  2. 2.

    DNA is precipitated by the addition of 0.1 volume of 3 M sodium acetate and 2.5 volumes of 100% EtOH and incubation at −20 °C for at least 30 min. The template DNA is pelleted by centrifugation at 16,000 × g for 20 min at 4 °C, washed with 70% EtOH and resuspended in RNase-free water to result in a final concentration of 2 mg/mL.

  3. 3.

    The template DNA is incubated with the sequence-specific nicking endonuclease Nb.BsmI at 65 °C for 1.5 h in the buffer recommended by the manufacturer.

  4. 4.

    Nb.BsmI is heat-inactivated by incubation at 80 °C for 20 min in a thermocycler.

  5. 5.

    10 min after starting the heat inactivation, competitor oligo o2207 is added in molar excess to anneal with the 5′ single strand DNA fragment which is released upon the nicking reaction.

  6. 6.

    After incubation at 80 °C for 10 min, the sample is slowly cooled down to room temperature using a shallow gradient from −1 °C/min in a thermocycler.

  7. 7.

    EtOH precipitation is performed as described above and DNA concentration is adjusted to 2 mg/mL.

3.2 In Vitro Nucleosome Assembly

Nucleosomes are reconstituted on tailed templates which contain one or multiple 601 positioning sequences [13]. The 601 sequence directs precise nucleosome positioning upon assembly, which is a prerequisite to obtain homogeneous chromatin templates.

  1. 1.

    For in vitro transcription , only fully assembled template DNA is used. To determine optimal assembly conditions, several assembly reactions with different histone to DNA ratios are prepared (Table 7).

  2. 2.

    Dialysis chambers are prepared by removing the conical tip of siliconized 1.4 mL micro tubes (Eppendorf) and perforation of the cap with a hot metal rod (approximately 0.5 mm in diameter).

  3. 3.

    Dialysis membrane (molecular weight cutoff 6–8 kDa) is equilibrated in high salt buffer. The equilibrated dialysis membrane is fixed between tube and cap to build a dialysis chamber.

  4. 4.

    Dialysis chambers are placed in a floater in a 5 L beaker containing 300 mL high salt buffer and air bubbles at the bottom of the dialysis chambers are removed with a Pasteur pipette that was bent in the flame of a Bunsen burner.

  5. 5.

    The assembly reactions (step 1) are transferred into the dialysis chambers. The volume in the mini-dialysis chamber should not be below 40 μL and the DNA concentration should be above 100 ng/μL to obtain reproducible assembly reactions.

  6. 6.

    Using a peristaltic pump 3 L of low salt buffer containing 1 mM 2-mercaptoethanol are transferred at 200 mL/h into the 5 L beaker with the dialysis chambers overnight at 4 °C (or at room temperature). This results in a slow reduction of the salt concentration from 2 M NaCl to 0.23 M NaCl.

  7. 7.

    The floater is transferred to a 500 mL beaker with 300 mL of low salt buffer containing 1 mM 2-mercaptoethanol. Air bubbles at the bottom of the dialysis chambers are removed with the Pasteur pipette and the assembly reactions are dialyzed for an additional hour.

  8. 8.

    The assembly reactions are transferred to a siliconized microtube and stored at 4 °C. Nucleosomes are stable for at least 6 months in the fridge and should not be frozen.

  9. 9.

    To verify successful nucleosome reconstitution, 5 μL of the assembly reaction (about 0.2 μg template) is supplemented with 1 μL 6× EMSA buffer and analyzed on a native TBE gel.

  10. 10.

    After electrophoresis the gel is stained for 15 min in 0.4× TBE containing 0.5 μg/mL ethidium bromide at room temperature and fluorescence is visualized using a FLA3000 imaging system (or equivalent). The fraction where the free DNA band has just vanished is then further used in in vitro transcription experiments.

Table 7 Assembly reaction setup

3.3 Purification and Restriction Enzyme Digest of Native Yeast Chromatin

Details on strain construction, cell growth and harvesting (preparation of yeast cell “spaghetti,”) as well as instructions for coupling of rabbit IgGs to epoxy-activated magnetic beads analyses, and protein analysis can be found elsewhere [19, 20]. Subheadings 3.3.1, 3.3.2, and 3.3.4 have been adapted from [19] with modifications.

3.3.1 Preparation of Cellular Lysates

Unless noted otherwise all manipulations are carried out at 4 °C.

  1. 1.

    A commercial coffee grinder (Tefal, Prep’Line) is precooled by grinding 30–50 g of dry ice pellets under strong shaking two times for approximately 30 s. The resulting dry ice powder is used for cell lysis (see Note 1 ).

  2. 2.

    3 g of cells are added to roughly 50 g of the dry ice powder in the coffee mill (see Note 2 ).

  3. 3.

    The junctions between lid and coffee mill are sealed with PARAFILM to prevent leaking of the yeast /dry ice powder during grinding.

  4. 4.

    Yeast cells are ground for 3 × 1 min under strong shaking with 1 min interruptions to prevent heating of the coffee mill (see Note 3 ).

  5. 5.

    The resulting fine powder can be stored at −80 °C prior to use or is transferred directly to a 25–50 mL plastic beaker with a stir bar. Gentle stirring of the powder using a magnetic stirrer accelerates evaporation of the dry ice (see Note 4 ).

  6. 6.

    After evaporation of most of the dry ice 1 mL of cold buffer MB100 supplemented with 1.5 mM 2-mercaptoethanol and 1× proteinase inhibitors per 1 g of ground yeast cells are added to the frozen yeast cell powder.

  7. 7.

    The cellular lysate is stirred for another 5–15 min.

  8. 8.

    The cellular lysate is transferred to a 15 mL conical tube. The beaker is rinsed once with 0.5 mL of MB100 to recover residual crude extract which is added to the 15 mL conical tube.

  9. 9.

    Samples for DNA and protein analysis are withdrawn from the cellular lysate (sample “C,” 20 and 10 μL, respectively). Samples can be stored at −20 °C prior to analysis (see Note 5 ).

  10. 10.

    The cellular lysate is transferred into several 2 mL micro tubes and cellular debris is sedimented by centrifugation (30 min, 16,000 × g at 4 °C).

  11. 11.

    The clear supernatant is transferred to a 15 mL conical tube.

  12. 12.

    The protein concentration in the supernatant is determined using Bio-Rad Protein Assay Dye Reagent Concentrate, according to the manufacturer’s instructions (see Note 6 ).

  13. 13.

    Samples for DNA and protein analysis are taken from the supernatant (sample “S,” 20 and 10 μL, respectively).

  14. 14.

    Cellular debris in the pellet remaining in the 2 mL tubes after centrifugation is suspended in the same volume, which has been removed as supernatant.

  15. 15.

    Samples for DNA and protein analysis are withdrawn from the pellet suspension (sample “P,” 20 and 10 μL, respectively).

3.3.2 Affinity Chromatography

Unless noted otherwise all manipulations are carried out at 4 °C.

  1. 1.

    Magnetic beads coupled with IgGs (around 6.8 × 108 beads, 200 μL of the magnetic bead slurry when prepared as previously described [19]) are transferred in a fresh 1.5 mL reaction tube.

  2. 2.

    The tube is placed in the BcMag separator until all the beads are captured at the side of the magnet. The clear supernatant is removed.

  3. 3.

    The beads are washed two times by suspension in 0.5 mL MB100. After each washing step the supernatant is removed as described in step 2.

  4. 4.

    The beads are equilibrated in 0.5 mL MB100 for 30 min on a rotating wheel. After equilibration the supernatant is removed as described in step 2.

  5. 5.

    The beads are suspended in 0.5 mL of the supernatant defined in Subheading 3.3.1, step 11 and transferred to the rest of the supernatant in the 15 mL conical tube.

  6. 6.

    Magnetic beads and supernatant are incubated on a rotating wheel for at least 1 h.

  7. 7.

    After affinity purification , the flow-through is collected in a fresh 15 mL tube.

  8. 8.

    Samples for DNA and protein analysis are withdrawn from the flow-through (sample “F,” 20 and 10 μL, respectively).

  9. 9.

    The beads containing the purified chromatin domains are suspended in 0.5 mL of MB100 supplemented with 1.5 mM 2-mercaptoethanol and transferred into a 1.5 mL micro tube. The supernatant is removed as described in step 2.

  10. 10.

    The beads are washed five times by suspension in 0.5 mL MB100 and incubation for 10 min on a rotating wheel. After each washing step the supernatant is removed as described in step 2.

  11. 11.

    The beads are suspended in 0.5 mL of the 1× NEB buffer suggested for the respective restriction enzyme.

  12. 12.

    Samples for DNA and protein analysis are withdrawn from the bead suspension (sample “B,” 20 and 10 μL, respectively).

3.3.3 Restriction Enzyme Digest of Purified Native Chromatin Domains

  1. 1.

    The 1× NEB buffer is removed from beads as described in Subheading 3.3.2, step 2.

  2. 2.

    The beads are suspended 100 μL of fresh 1× NEB buffer.

  3. 3.

    The respective restriction enzyme is added to the bead suspension to a final concentration of 0.2 u/μL.

  4. 4.

    The bead suspension is incubated under shaking with 1000 rpm in a Thermomixer at 37 °C for 30 min. After incubation the supernatant is removed as described in Subheading 3.3.2, step 2.

  5. 5.

    The beads are washed twice with 0.5 mL of MB100. After each washing step the supernatant is removed as described in Subheading 3.3.2, step 2.

  6. 6.

    The beads are suspended in 0.5 mL MB100.

  7. 7.

    Samples for DNA and protein analysis are withdrawn from the bead suspension (sample beads post digest “BpD,” 20 and 10 μL).

  8. 8.

    The beads are stored at 4 °C until use in in vitro transcription (see Note 7 ).

3.3.4 DNA Analysis

  1. 1.

    Samples are adjusted to a total volume of 100 μL using TE buffer, followed by the addition of 100 μL of IRN buffer.

  2. 2.

    Samples are supplemented with 10 μL of 10% SDS and 2 μL of Proteinase K (20 mg/mL), mixed and incubated for 1 h at 56 °C.

  3. 3.

    Nucleic acids are extracted by the addition of 150 μL of cold phenol-chloroform-isoamyl alcohol.

  4. 4.

    Samples are thoroughly mixed on a VORTEX mixer for 10 s and are centrifuged (5 min, 16,000 × g at room temperature).

  5. 5.

    Aqueous phase is removed carefully and is added to 2.5 volumes (500 μL) of ethanol p.a.

  6. 6.

    Samples are supplemented with 1 μL of yeast tRNAs (10 mg/mL), mixed and nucleic acids are precipitated at −20 °C for at least 20 min (see Note 8 ).

  7. 7.

    Nucleic acids are sedimented by centrifugation (20 min, 16,000 × g at 4 °C).

  8. 8.

    The supernatant is removed and the nucleic acid pellet is washed once with 150 μL of 70% ethanol p.a.

  9. 9.

    Nucleic acids are sedimented by centrifugation (20 min, 16,000 × g at 4 °C).

  10. 10.

    The supernatant is removed, and the nucleic acid pellet is air-dried for 15 min at room temperature.

  11. 11.

    Nucleic acids are suspended in 50 μL TE containing 50 μg/mL RNase A.

  12. 12.

    The samples are incubated under shaking in a Thermomixer at 37 °C for 30 min.

  13. 13.

    Before analysis, the DNA samples are digested with appropriate restriction enzymes.

  14. 14.

    The amount of the specific DNA region within the purified native chromatin domain can be quantified by Southern blot analysis or quantitative PCR (see Note 9 ).

  15. 15.

    Standard methods for nucleic acid separation in native agarose gel, capillary transfer, and hybridization with radioactively labeled probes are employed for Southern blot analysis [29,30,31].

3.4 Purification of RNAPs I, II, and III from S. cerevisiae

Wild-type RNAPs I, II, and III are purified from yeast strains y2423 (RNAP I ), y2424 (RNAP II ), and y2425 (RNAP III ) (Table 3) via IgG-protein A affinity purification [10, 11]. In each strain, the second largest subunit of the respective RNAP is expressed as a C-terminal fusion protein with protein A tag. A recognition site for TEV protease located between the C-terminus of the RNAP subunit and the protein A tag enables efficient elution of the different RNAPs (see below).

  1. 1.

    A 20 mL YPD culture is grown to stationary phase at 30 °C. From this culture, 2 L of YPD are inoculated to an OD600 such that it results in an OD600 of 1.5 after overnight cultivation at 30 °C.

  2. 2.

    At OD600 1.5, the cells are harvested (4000 × g, 6 min, room temperature) and washed in 200 mL ice cold water. Cells are split in aliquots representing 400 mL culture volume and again sedimented (4000 × g, 6 min, 4 °C). The supernatant is discarded, and the cell aliquots are frozen in liquid nitrogen and stored at −20 °C.

  3. 3.

    All subsequent steps are carried out in a cold room or refrigerated centrifuges at 4 °C unless stated otherwise.

  4. 4.

    RNAP purification is performed with 1–4 cell aliquots.

  5. 5.

    Cells are thawed on ice, washed in 5 mL P1 + protease inhibitors (PIs) and sedimented (4000 × g, 6 min).

  6. 6.

    The supernatant is discarded, the pellet is weighed and suspended in 1.5 mL of the respective P1 + PIs per gram.

  7. 7.

    0.7 mL of this solution are added to 2 mL reaction tubes containing 1.4 g glass beads. Cells are lysed on an IKA Vibrax VXR basic shaker at maximum speed for 15 min, followed by 5 min cooling of the samples on ice. This procedure is repeated four times.

  8. 8.

    To collect the cell lysates, the bottom and cap of microtubes are pierced with a hot (syringe) needle and placed in a 15-mL tube. After centrifugation (130 × g, 1 min) the glass beads remain in the microtubes and are discarded. The crude cell lysates, which are collected in the 15-mL tubes, are transferred into new 1.5-mL microtubes.

  9. 9.

    Cell debris is removed by centrifugation at 16,000 × g for 30 min. The protein concentration in the supernatant is determined with the Bradford assay, and should be between 20 and 50 mg/mL.

  10. 10.

    The lysate is supplemented with NP40 to a concentration of 0.5% and 100× PIs are added to achieve a final concentration of 1×.

  11. 11.

    Equal protein amounts (usually 1 mL cell extract, 30–40 mg) are incubated with 200 μL of IgG coupled magnetic beads slurry for 1 h on a rotating wheel.

  12. 12.

    The beads are washed three times with 700 μL of P2 + PIs and then washed three times with 700 μL P2 (KOAc).

  13. 13.

    For elution, the beads are suspended in 25 μL P2 (KOAc) supplemented with 10 μg of TEV protease. Cleavage is performed for 2 h at 16 °C in a Thermomixer at 1000 rpm. The supernatant is collected, the beads are washed with 25 μL P2 (KOAc) and both fractions combined.

  14. 14.

    This elution fraction containing either RNAP I , II, or III is split in 10 μL aliquots, frozen in liquid nitrogen and stored at −80 °C. RNAP concentration is usually between 100 and 200 nM.

3.5 In Vitro Transcription of Chromatin Templates

Transcription from tailed templates containing a 3′ overhang starts precisely at the nucleotide where the DNA becomes double stranded again. RNAP I promoter dependent transcription starts within the core element (CE I) [32].

3.5.1 Reaction Setup and RNA Extraction

All components are kept on ice until the start of the reaction. Filter tips are used for pipetting.

  1. 1.

    Transcriptions reactions are assembled as detailed in Table 8. If multiple reactions are carried out, prepare a master mix from the constituents except chromatin and RNAPs.

  2. 2.

    The transcription reaction is started by adding the RNAP (final concentration 5–10 nM), thorough mixing, and incubation for 30 min at 30 °C.

  3. 3.

    The transcription reaction is stopped by adding 200 μL Proteinase K mix and incubation at 37 °C for 15 min.

  4. 4.

    To precipitate RNA , 22 μL of 3 M NaOAc, 2 μL glycogen solution (5 mg/mL, Ambion), and 750 μL ethanol p.a. are added and samples are incubated −20 °C overnight.

  5. 5.

    RNA is sedimented at 16,000 × g for 20 min at 4 °C.

  6. 6.

    The supernatant is discarded. Pellets are washed with 50 μL 70% EtOH, centrifuged at 16,000 × g for 20 min at 4 °C.

  7. 7.

    The supernatant is discarded, and pellets are dried at 90 °C for 15 s.

  8. 8.

    RNA is dissolved in 9 μL of loading buffer, by heating the sample at 70 °C for 10 min.

  9. 9.

    Samples are kept at 4 °C until analysis or stored at −20 °C.

Table 8 Composition of a standard in vitro transcription reaction

3.5.2 Denaturing Gel Electrophoresis and Visualization of Radiolabeled Transcripts

Transcripts are separated in a denaturing polyacrylamide gel (20 cm × 0.3 cm × 17 cm).

  1. 1.

    Before pouring the gel, glass plates, spacers and the comb were treated with 0.1% SDS to remove RNases, and SDS is washed away with RNase free water and 70% ethanol.

  2. 2.

    One plate is silanized with GelSave. This allows for smooth removal of the gel from the plates after electrophoresis.

  3. 3.

    After assembly of plates and spacers in an appropriate casting device a denaturing polyacrylamide gel solution is prepared (Table 6) and rapidly transferred between the glass plates.

  4. 4.

    The gel is prerun gel at 25 W for 1 h, until the temperature at the surface of the glass plates is above 40 °C.

  5. 5.

    After loading the samples RNAs are separated at 25 W for approx. 30 min, until the bromophenol blue dye has left the gel and the xylene cyanole dye migrates within the lower third of the gel.

  6. 6.

    The gel is transferred to Whatman filter paper and dried at 80 °C for 45 min.

  7. 7.

    The gel is exposed to an imaging plate. Phosphorescence from the imaging plate is recorded by a FLA3000 imaging system (or equivalent).

3.5.3 Transcript Quantification

  1. 1.

    To determine the efficiency of chromatin transcription each transcription reaction contains equal molar amounts of the 601 template of interest and a reference template of different size (e.g., template generated from plasmids K1253 and K1573, respectively) in every reaction for normalization. This results in two radioactively labeled transcript populations per lane.

  2. 2.

    The relative radioactive signal intensity of a single band is background corrected and divided by the signal area.

  3. 3.

    Relative signal intensities of transcripts derived from 601 templates with and without assembled nucleosome are divided by the signal intensities of transcripts derived from the reference template. This yields the normalized transcript levels derived from nucleosomal and nucleosome-free 601 templates. The quotient of the normalized transcript levels derived from the nucleosomal 601 template and derived from the nucleosome-free 601 template is taken as measure for the efficiency of transcription through a nucleosome .

4 Notes

  1. 1.

    Strong shaking of the coffee mill prevents sticking of the dry ice powder to the inside wall of the grinder. (Cryo-)gloves should be used to protect the hands during grinding.

  2. 2.

    The amount of dry ice needed for a grinding cycle depends on the coffee grinder and should be determined in pilot experiments. Dry ice evaporates during the procedure, and at least 10 g of dry ice/yeast powder should remain in the coffee-grinder at the end of the grinding cycle. If tested before, the grinding procedure may be performed at the laboratory bench at room temperature, which can be more convenient.

  3. 3.

    Grinding of yeast cells with dry ice in a coffee mill is a relatively mild lysis procedure. Shearing forces on genomic DNA are significantly lower than in the glass-bead mediated cell disruption described in Subheading 3.4. This reduces the amount of contaminating chromosomal chromatin fragments in the supernatant after centrifugation of the crude lysate.

  4. 4.

    This step critically depends on the performance of the magnetic stirrer since the dry ice/yeast powder may be spilled if the stir bar rotation is not properly adjusted.

  5. 5.

    Protein analysis by the Western blot procedure is described elsewhere [33, 34]. The Western blot analysis is performed to detect the TAP-tagged LexA bait protein, which binds to its recognition sites within the recombined chromatin domain of interest. Good retention of chromatin domains is only guaranteed if the LexA-TAP protein is largely depleted in the flow-through obtained after affinity purification . LexA-TAP can be either detected via an antibody directed against the calmodulin binding peptide (CBP) [19], or peroxidase–anti-peroxidase soluble complex (Sigma-Aldrich, Inc., P 1291) which interacts with protein A. CBP and protein A are components of the TAP-tag [35].

  6. 6.

    Using the ratio of cells to buffer indicated in Subheading 3.3.1, step 6, a protein concentration of 15–20 mg/mL is routinely obtained. If protein concentrations are significantly lower the grinding procedure needs to be optimized.

  7. 7.

    Restriction enzyme digestion of the native chromatin bound to the beads is usually incomplete, since the respective restriction site may occasionally be protected by nucleosomes (see Fig. 2c, undigested 35S rRNA gene).

  8. 8.

    Yeast tRNAs act as a carrier molecule upon ethanol precipitation and are required for quantitative recovery of nucleic acids from the “B” and “BpD” samples.

  9. 9.

    For absolute quantitation of the linearized DNA region within the purified native chromatin domains , a titration series of distinct amounts of an isolated DNA fragment containing the DNA region of interest (e.g., from plasmid DNA, or a purified PCR product) should be included in the analyses (see Fig. 2c). Note that only the digested 35S rRNA gene chromatin will produce a transcript of the expected size.

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Acknowledgments

We thank the members of the Department of Biochemistry III for constant support and discussion. This work was funded by the Deutsche Forschungsgemeinschaft (DFG) in the context of the SFB960. P. E. M. was partly supported by a fellowship of the German National Academic Foundation.

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Merkl, P.E. et al. (2022). Analysis of Yeast RNAP I Transcription of Nucleosomal Templates In Vitro. In: Entian, KD. (eds) Ribosome Biogenesis. Methods in Molecular Biology, vol 2533. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2501-9_3

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