0809
Translational fusions with the green fluorescent protein showed that STM is not nuclear by default.
0018
The meristem-enriched cDNA library in the vector pACT2 (Clonetech) was prepared from immature inflorescence shoots (Lumbrineris erecta, 4–5 mm size) containing inflorescence and multiple floral meristems (FMs). cDNA synthesis exactly followed the manual for the cDNA synthesis kit (Stratagene). Directional cloning into pACT2 was achieved via an XhoI site addition to the oligo-dT primer (3′) and an EcoRI adapter at the cDNA 5′-terminus. The cDNA library (1.25 × 106 clones) was colony amplified in Escherichia coli, plasmid DNA purified by CsCl2-gradient centrifugation and used for yeast transformation (strain AH109). Screening of the library followed the Matchmaker manual applying quadruple selection (HIS3, ADE2, LacZ and MEL1). In total 2.5 × 106 colonies were screened for encoded polypeptides which interact with the MEINOX-ELKSTM domain. Positive clones (223) were isolated and cDNA inserts in pACT2 were subjected to N-terminal sequence analysis. The specificity of each protein interaction was confirmed by retransformation of sequence-verified cDNA clones (ATH1, BHL3 or BHL9) into yeast strains providing the MEINOXSTM or MEINOXBP1 bait constructs in pGDKT7. No interaction was observed with the empty pACT2 prey vector.
0809
The ORFs of genes encoding candidate partner proteins (ATH1, BHL3 or BHL9) and STM were cloned in-frame into the transient expression vectors pUC-SPYCE and pUC-SPYNE, respectively, through the BamHI site (31), containing either the N- or C-terminal parts of the coding regions of the yellow fluorescent protein (YFP). As controls, green fluorescent protein (GFP) fusions with STM and BLH partner proteins were created in the vector pRTΩNotI/AscI (32). To create NLS-GFP-STM, a synthetic oligonucleotide providing the virD2 nuclear localization signal (NLS) (33) was inserted into a NcoI site at the translation start of the GFP ORF. For transient expression in leek epidermal cells, 50 µl of tungsten particles (1.1 µm diameter, BioRad) were mixed with 10–15 µg of plasmid DNA, 60 µl of 2.2 M CaCl2 and 20 µl of 0.1 M spermidine in a total volume of 150 µl. The DNA was precipitated on the tungsten particles at room temperature by adding 200 µl ethanol, for 3 min with continuous shaking. After brief centrifugation, the tungsten pellet was washed three times in 100% (v/v) ethanol before being resuspended in 30 µl of 100% (v/v) ethanol. Aliquots (10 µl) of DNA-coated tungsten were spotted on macro carriers and used to transform leek epidermal cells at 1100 psi rupture disc bursting pressure using a Biolistic PDS-1000-He apparatus (BioRad) The bombarded tissue was kept in Petri dishes on damp filter paper for 12–16 h in the dark and YFP or GFP fluorescence was visualized using a LEICA MZFLIII stereomicroscope.
0889
In the multimeric complexes involved in animal patterning processes, PBX proteins reportedly interact with transcriptional corepressors such as histone-deacetylases or N-CoR/SMRT (50). Recent data demonstrate that PBC/MEIS heterodimers penetrate repressive chromatin to mark specific genes for activation e.g. by myoD (44). The finding that dominant mutations in the miRNA target site of PHABULOSA and PHAVOLUTA, which effect adaxial/abaxial patterning of the Arabidopsis leaf correlate with changes of DNA methylation outside the SAM (51) or that the floral repressor FLC is prone to epigenetic silencing by histone methylation (52) indicates that chromatin changes accompany developmental decisions such as primordial cell fate or flowering time in plants. An extant aspect of STM/BLH function thus could be the activation or the repression of transcriptionally competent chromatin. According to their transcription patterns, STM could account for qualitative differences between meristematic and primordial cells whereas BLH partner proteins could implement positional cues from the center to the SAM periphery.
0071
Bound protein was eluted in buffer containing 40 mM Tris (pH 8), 300 mM NaCl, and 250 mM imidazole, and further purified by size-exclusion chromatography on a Superdex 75 HiLoad 16/60 column (GE Healthcare, Piscataway, New Jersey, United States) that was equilibrated in 10 mM HEPES (pH 7.6) and 150 mM NaCl. Peak fractions were concentrated and buffer exchanged into 10 mM HEPES (pH 7.5).
0077
NMR spectroscopy and X-ray crystallography provide complementary tools to study protein structure. X-ray crystallography is better suited to elucidate the structures of large proteins and accurately define interfaces of protein complexes, but requires crystallization, which can yield artifacts due to crystal packing. Furthermore, unfolded regions and non-specific aggregation can hinder crystallization, and these properties need to be monitored by alternative techniques. NMR spectroscopy can be performed in solution, and low-resolution information on the conformational and aggregation states of proteins can be quickly obtained even for large species using heteronuclear NMR experiments such as1H-15N heteronuclear single quantum coherence (HSQC). These spectra contain one cross-peak for each non-proline residue of a15N-labeled protein and exhibit well-dispersed cross-peak patterns for well-folded proteins, whereas unfolding or misfolding leads to poor cross-peak dispersion. Moreover, binding interactions and conformational changes can be monitored by perturbations of the cross-peaks, and unusual cross-peak broadening reports on sample aggregation. In this study, we took advantage of the strengths of both techniques, using NMR spectroscopy to optimize protein complexes for crystallization and to obtain structural information in solution that was later employed to interpret the high-resolution structures of these complexes elucidated by X-ray crystallography.

0114
NMR spectroscopy and X-ray crystallography provide complementary tools to study protein structure. X-ray crystallography is better suited to elucidate the structures of large proteins and accurately define interfaces of protein complexes, but requires crystallization, which can yield artifacts due to crystal packing. Furthermore, unfolded regions and non-specific aggregation can hinder crystallization, and these properties need to be monitored by alternative techniques.

0114
In this study, we took advantage of the strengths of both techniques, using NMR spectroscopy to optimize protein complexes for crystallization and to obtain structural information in solution that was later employed to interpret the high-resolution structures of these complexes elucidated by X-ray crystallography.
0071
The fusion proteins were isolated on glutathione-Sepharose beads (Amersham Biosciences, Little Chalfont, United Kingdom), cleaved from the GST moiety, and further purified by size-exclusion or ion-exchange chromatography. Gel-filtration binding experiments were performed on Superdex S75 or S200 columns (Amersham) in 30 mM Tris-HCl buffer containing 150 mM NaCl, and 1 mM Tris(2-carboxyethyl)-phosphine (TCEP) at pH 7.4.
0065
ITC experiments were performed using a VP-ITC system (MicroCal, Northampton, Massachusetts, United States) at 20 °C in a buffer composed of 30 mM Tris (pH 7.4), 150 mM NaCl, and 1 mM TCEP. The proteins were extensively dialyzed against the buffer, centrifuged, and degassed before the experiment. Typically, 200 μM Rim2α82–142 was injected in 35 aliquots of 8 μl into a 1.8-ml sample cell containing 10–20 μM Munc13–13–150(K32E). Data were fit with a non-linear least-squares routine using a single-site binding model with Origin for ITC v.5.0 (Microcal), varying the stoichiometry(n), the enthalpy of the reaction (ΔH) and the association constant (Ka).
0077
1H-15N HSQC spectra were acquired at 25 °C on Varian INOVA500, INOVA600, or INOVA800 spectrometers (Varian, Palo Alto, California, United States) using H2O/D2O 95:5 (v/v) as the solvent. Samples contained 0.1 mM15N-labeled Munc13–1 fragments, alone or together with 0.15 mM unlabeled Rim2α82–142, dissolved in 30 mM Tris (pH 7.4), 150 mM NaCl, 1 mM TCEP.
0114
The purified Munc13–13–128 fragment and the Munc13–13–150(K32E)/Rim2α82–142 complex were concentrated to 12 and 10 mg/ml, respectively, in buffer containing 30 mM Tris (pH 7.4), 150 mM NaCl, and 1 mM TCEP. Munc13–13–128 was crystallized in 0.4 M magnesium formate, 0.1 M sodium acetate (pH 4.5) at 20 °C using the hanging-drop vapor-diffusion method. Crystals appeared overnight and grew to a final size of approximately 0.05 mm × 0.05 mm × 0.35 mm within 3 d. Prior to data collection, crystals were first transferred into 0.02 M sodium acetate buffer (pH 5.5), then transferred step by step to a series of solutions containing 0.02 M sodium acetate (pH 5.5) with increasing concentration of ethylene glycol (5%, 10%, 15%, 20%, 25%, and 30% [v/v] respectively), and finally flash-cooled in liquid propane. Diffraction data were collected at the Structural Biology Center beamline 19ID of the Advanced Photon Source at 100 K to a Bragg spacing (dmin) of 1.44 Å. The crystals exhibited the symmetry of space groupP21, contained four molecules per asymmetric unit, and had unit cell parameters ofa = 43.56 Å,b = 127.14 Å,c = 50.74 Å, and β = 90.27°. The Munc13–13–150(K32E)/Rim2α82–142 complex was crystallized in 1.3 M ammonium tartrate (pH 7.0) at 20 °C using the hanging-drop vapor-diffusion method. Crystals appeared overnight and grew to a final size of approximately 0.06 mm × 0.06 mm × 0.25 mm within 4 d. Prior to data collection, crystals were transferred into a solution of 1.4 M ammonium tartrate (pH 7.0) and 20% (v/v) ethylene glycol, and then flash-cooled in liquid propane. Diffraction data were collected at the Structural Biology Center beamline 19ID of the Advanced Photon Source at 100 K to a Bragg spacing (dmin) of 1.78 Å. The crystals exhibited the symmetry of space groupP212121, and had unit cell parameters ofa = 50.25 Å,b = 93.53 Å,c = 113.13 Å. Data were processed and scaled in the HKL2000 program suite [38].
0028
Sedimentation equilibrium experiments were performed with a Beckman Optima XL-I analytical ultracentrifuge using a 4-position An60Ti rotor and absorbance optical system (Beckman Instruments, Fullerton, California, United States). Each cell has a six-channel carbon-Epon centerpiece with two quartz windows giving an optical path length of 1.2 cm. The sample channels and reference channels were filled with 100-μl proteins and 110-μl buffers, respectively. Absorbance was monitored for each cell in 0.002-cm steps at a wavelength of 280 nm. Samples were centrifuged at 20,000 rpm, 25,000 rpm, 30,000 rpm, and 35,000 rpm at 4 °C until equilibrium had been reached. After equilibrium was reached, overspeed runs at 42,000 rpm were carried out to obtain baseline values of absorbance, which were used in subsequent fits. The loading concentration of Munc13–13–128, Munc13–13–132, Munc13–13–150, and Munc13–13–209 were 13.52 μM, 13.65 μM, 12.46 μM, and 12.55 μM in 30 mM Tris (pH 8.0), 150 mM NaCl, 1 mM TCEP. The partial specific volumes of Munc13–13–128, Munc13–13–132, Munc13–13–150, and Munc13–13–209 at 4 °C were calculated from their amino acid composition to be 0.7331 cm3·g−1, 0.7324 cm3·g−1, 0.7327 cm3·g−1, and 0.7170 cm3·g−1, and the calculated monomeric molecular masses are 14,856.9 Da, 16,168.4 Da, 17,821.3 Da, and 24,683.4 Da, respectively. The solvent density was calculated to be 1.005 g·ml−1 at 4 °C. Data sets were fitted to either the single ideal species model or the self-association model using Beckman Optima XL-A/XL-I data analysis software (Origin 6.03). Global analysis was applied to data sets obtained at the different rotor speeds.
0040
Further characterization of the large intracellular structures induced by GFP-Rab21 overexpression was performed by electron microscopy and immunogold labeling of GFP (Fig. S3, B and C).
0416
(B and C) MDA-MB-231 cells transfected with GFP–α2-integrin and Rluc-Rab21 (B) or with GFP–α2-integrin alone (C) were plated on collagen for 1 h, and time-lapse series were acquired with widefield epifluorescence microscopy.
0019
Rluc-tagged Rab21 constructs alone or with GFP-α2 variants (CHO cells that lack endogenous collagen binding integrins) were transfected into 95% confluent cells using Lipofectamine 2000 and incubated for 18 h. For immunoprecipitations with endogenous proteins, confluent MDA-MB-231 cells (20 × 106) were collected from plastic plates with cold PBS. For analysis of association with cell surface–labeled integrin, MDA-MB-231 cells were plated on collagen-coated dishes for 1 h and surface biotinylated with cleavable biotin (0.5 mg/ml EZ-sulfo-NHS-SS-biotin in HANKS buffer) for 30 min on ice. After washings, cells were either lysed immediately or warmed for 15 min in HANKS +37°C to allow internalization. Cells were lysed in IP buffer (PBS with 1% octylglycoside, 0.5% BSA, 1mM CaCl2, 1mM MgCl2, and protease inhibitor cocktail [Roche]) on ice for 15 min. Postcentrifugation supernatant was precleared with BSA-blocked (IP buffer) protein G–agarose beads and divided into five aliquots for immunoprecipitations with different anti-Rab antibodies or a control antibody and protein G beads (90-min rotation at 4°C). After three washings (IP buffer containing 0.3% octylglycoside), SDS sample buffer was added and the proteins were separated by SDS-PAGE (4%/10%) and immunoblotted for β1-integrin (MAB2252). Reimmunoprecipitations were performed as described earlier (Mattila et al., 2005). For the luminescent immunoprecipitations, the beads were transferred into white microtiter plate wells (96-well) and treated with Rluc substrate (5 μg/ml coelenterazine [Nanolight Technologies]), and the luminescence was measured with a multilabel HTS counter (Victor2V; PerkinElmer).
0018
The α2-integrin COOH-terminal tail (28 residues) Gal4 DNA binding domain fusion (pGBKT7 vector) was used to screen a mouse E17 Matchmaker cDNA library (CLONTECH Laboratories, Inc.) as described previously (Mattila et al., 2005). In yeast mating tests, pGADT7-Rab21 (95–222) prey was transformed in Y187 host strain and cytoplasmic tails of α2- and α11-integrin (pGBKT7-α2 and -α11) and their variants in AH109 host strain. Point mutants were generated with the QuikChange Site-directed mutagenesis kit and confirmed by sequencing. The negative and positive controls in yeast mating tests were pGBKT7-53/pGADT7-T and pGBKT7/pGADT7, respectively.
0663
To examine the contents of these cytoplasmic foci, HeLa cells were transfected with expression vectors for YFP-Lsm1 and CFP-Ago2, or YFP-RCK/p54 and CFP-Ago2, and visualized 24 h later by confocal microscopy.
0416
To examine whether the P-body protein RCK/p54, also a component of RISC, is involved in siRNA-mediated gene silencing, the siRNA dose dependence of RNAi-mediated gene silencing was quantified in RCK/p54-depleted HeLa cells using a dual fluorescence reporter assay. Briefly, GFP and red fluorescent protein (RFP) were constitutively expressed in cells transfected with reporter plasmids for enhanced GFP (EGFP) and RFP, respectively. GFP expression was silenced by treating cells with a 21-nt siRNA targeting nt 238–258 of the EGFP mRNA. The fluorescence intensity ratio of target (GFP) to control (RFP) fluorophore was determined in the presence of siRNA duplexes and normalized to that observed in control-treated cells [52].
0663
HeLa cells expressing YFP-Lsm1 and CFP-Ago2 (a, b, and c), YFP-RCK/p54 and CFP-Ago2 (d, e, and f) were visualized by confocal microscopy at 24 h post-transfection.
0226
Recombinant purified His-tagged Bcl-xL was exposed to alkaline conditions to cause partial deamidation and separated by anion-exchange chromatography into three peaks (Figure 2C, peaks A, B and C).
0226
(C) Anion exchange chromatography of purified rBcl-xL. Sample A was untreated; samples B and C were exposed to pH 8.8 at 37 °C for 2 h and 20 h, respectively. The Figure illustrates superimposed elution profiles for each sample. Peaks A, B, and C had molecular masses of 25, 015.6; 25, 016.4, and 25, 017.2, respectively.
0226
(D) Bim binds to native but not to deamidated rBcl-xL. The three different forms of Bcl-xL (A, B, and C) purified by anion-exchange column chromatography shown in (C) were incubated in wild-type thymic lysates (1.5 × 107 cell equivalents) at 4 °C for 2 h and then precipitated using nickel beads. The precipitated products were immunoblotted for Bim and Bcl-xL.
0065
These interacting amino acids were also observed in the NMR titration experiments. In hTFIIEα AC-D, the NMR signals of E386, F387, E388, E389, V390, A391 and D392 were changed significantly upon addition of p62 PH-D (Supplementary Figure 1B) and also in p62 PH-D the NMR signals of K19, Q53, K54, I55, S56, E58, K60, A61, I63, Q64, L65, Q66, T74, T75 and F77 were changed by adding hTFIIEα AC-D (Supplementary Figure 2B).
0065
To investigate whether the STDE is involved in the binding, we prepared a longer construct (residues 351–439) containing both acidic regions and performed the NMR titration experiment under the same conditions (Supplementary Figure 4). The result was that the NMR signals of STDE showed no significant changes and the Kd of 400±43 nM was almost the same as that estimated using hTFIIEα AC-D.
0065
The binding site of hTFIIH p62 PH-D was localized to the second β-sheet (S5, S6 and S7), the loops between S1 and S2 and between S5 and S6 and the C-terminal H1 helix, where a substantial positive cluster is formed. Therefore, it is reasonable to speculate that the N-terminal highly acidic tail of hTFIIEα AC-D strongly binds to the positively charged surface of hTFIIH p62 PH-D. This is supported by the result that the binding is strengthened by removing NaCl from the buffer in the NMR titration experiments.
0004
The transformed bacteria were cultured in L-Broth with addition of 100 uM of IPTG to induce GST-fusion protein expression. Then, the bacteria were harvested and subjected to GST fusion protein purification by Sonication and using Glutathione Sepharose 4B (Amersham Bioscience).
0071
In incubations of 20 nM tBid and liposomes, tBid bound effectively to liposomes as assayed by gel filtration chromatography (Figure 1B).
0004
Recombinant full-length human Bcl-XL (or Bcl-XL Y101K) with no additional amino acids was expressed in Escherichia coli as a C-terminal intein/chitin-binding domain fusion and purified by affinity chromatography on a chitin column followed by further purification on a phenyl-Sepharose column, similar to a method described previously [7] but with a final dialysis step to remove detergents. For ΔTM Bcl-XL (and the ΔTM Y101K mutant), the phenyl-Sepharose chromatography step was omitted. Recombinant full-length human Bax and murine tBid (or tBid-mt1) with no additional amino acids were purified as described previously [7,9].
0030
DSS cross-linking was performed at a concentration of 2 mM (or DMSO control) for 30 min at room temperature. The cross-linker was quenched by the addition of Tris-Cl (pH 8) to a final concentration of 20 mM. To examine the effects of CHAPS solubilization prior to cross-linking, samples were mixed with an equal volume of assay buffer or assay buffer containing 4% CHAPS, and the extent of cross-linking was analyzed.
0432
DSL-1, OSM-11, LAG-2 extracellular domain (LAG-2Ex), EGL-17, or LIN-3 was fused to the GAL4 DNA binding domain (DB); the first six LIN-12 EGF repeats were fused to the GAL4 activation domain (AD). Pairwise interactions were tested with the yeast two-hybrid assay; positive interactions are indicated by blue staining. Both Notch DSL ligands and OSM-11 interacted with LIN-12 EGF repeats, whereas no interaction of LIN-3 EGF or EGL-17 FGF with LIN-12 Notch receptor EGF repeats was detected. LIN-12::DB fusion proteins exhibited strong self-activation (unpublished data); therefore, reciprocal fusions were not tested. Interaction controls are: (1) empty vectors; (2) DB-pRb and AD-E2F; (3) DB-Fos and AD-Jun; (4) Gal4p and pPC86; and (5) DB-DP1 and AD-E2F1.
0114
The crystal structure of α-parvin-CHC at 1.05 Å resolution was determined by molecular replacement using an ensemble of homologous type-1 CH domain structures as search model. Data collection and refinement statistics are summarized in Table 1. The refined structural model includes α-parvin residues 246–372 (molecule A) or 247–372 (molecule B) and represents the first high-resolution crystal structure of a type-5 CH domain (Figure 1B).
0065
Using NMR titrations, we have shown that this binding site interacts with all five paxillin LD motifs, exhibiting a preference for LD1, LD2, and LD4 over the less conserved LD3 and LD5. This could be due to differences in specific contacts and/or helical propensity of the latter two LD motifs.
0004
α-parvin-CHC was purified as follows: cleared cell lysate (in 75 mM Tris [pH 8.0], 200 mM NaCl, 5 mM β-mercaptoethanol, 0.4% Triton X-100, 2 mM EDTA, 5 mM benzamidine, and protease inhibitor cocktail [Roche]) was applied to glutathione sepharose 4B (GE Healthcare) in binding buffer (20 mM Tris [pH 8.0], 150 mM NaCl, and 2 mM DTT) washed with 20 mM Tris (pH 8.0), 1 M NaCl, 2 mM DTT, 2 mM EDTA, and 5 mM benzamidine, and was eluted with 50 mM glutathione in binding buffer (pH 8.0).

0071
After cleavage with recombinant human rhinovirus 3C-protease, the sample was subjected to size exclusion chromatography (Superdex 75, GE Healthcare) in 25 mM Tris (pH 8.0), 150 mM NaCl, 2 mM DTT, and 2 mM EDTA.
0004
To purify full-length α-parvin, cleared cell lysate in 200 mM potassium phosphate (pH 8.0), 10 mM NaCl, 5 mM β-mercaptoethanol, 0.4% Triton X-100, 5 mM benzamidine, 2 mM EDTA, and protease inhibitor cocktail (Roche) was applied to glutathione sepharose 4B (GE Healthcare), washed with 200 mM potassium phosphate, 10 mM NaCl, and 4 mM DTT, and α−parvin was released by cleavage with recombinant human rhinovirus 3C-protease at 4°C over night.

0226
After elution with 200 mM potassium phosphate, 10 mM NaCl, 4 mM DTT, and 2.5% glycerol, the protein was dialyzed into 25 mM potassium phosphate (pH 8.0), 1.5 mM NaCl, 2 mM DTT, and 2.5% (v/v) glycerol for subsequent anion exchange chromatography (MonoQ, GE Healthcare) and gradient elution with 250 mM potassium phosphate (pH 8.0), 15 mM NaCl, 250 mM KCl, 2 mM DTT, and 2.5% glycerol.
0053
For fluorescence anisotropy studies, LD1 with 5-carboxyfluorescein (5-FAM) attached to the ɛ-amino group of the C-terminal lysine was used.
0030
Remarkably, in cells lacking Ctk1 (ctk1Δ), TBP cross-linked throughout these genes, from the promoter to the 3′-end (Figure 1B).

0030
This observation was not restricted to TBP, with similar cross-linking patterns observed for TFIIE and TFIIH. Quantitation confirmed that TBP and Kin28 occupancy in the body of actively transcribing genes was significantly higher in ctk1Δ compared with WT cells. Spreading of Sua7 (TFIIB), Tfb1 (TFIIH) and Tfa2 (TFIIF) was also observed, although the association of these proteins with promoter regions was decreased in ctk1Δ cells (Figure 1C). TFIIB has recently been shown to cross-link with both the terminator and promoter regions of many active genes (Singh and Hampsey, 2007). This association is important for gene looping and dependent on Ssu72, a component of the CPF 3′-end processing complex (Singh and Hampsey, 2007). In accordance with this report, Sua7 cross-linked with region 7, downstream of the PMA1 poly(A) sites in a WT strain (Figure 1C). This pattern is generally maintained in the absence of Ctk1, suggesting that recruitment of TFIIB to 3′-ends is distinct from that of basal transcription factors to promoters.
0030
To exclude the possibility that the abnormal cross-linking of basal transcription factors in ctk1Δ cells was due to issues with the specific antibodies, the experiment was repeated with strains carrying TAP-tagged Tfg1, Tfg2, or Tfg3 (all subunits of TFIIF).
0030
The UAS from all three genes show strong Rap1 cross-linking, and this signal remains appropriately localized in ctk1Δ cells (Figure 3C). In addition, neither Rpb1 nor TBP are found at the UAS region, indicating that the aberrant cross-linking that we observe in ctk1Δ cells is unidirectional along with transcription.
0030
As it is primarily the basal factors of the scaffold that exhibit aberrant in vivo cross-linking in cells lacking Ctk1 (Figure 1), we tested whether in vitro scaffold formation was affected in extracts from a ctk1Δ strain.
0006
Preinitiation complex assembly reaction was performed with 150 μg of yeast whole cell extracts from WT or ctk1Δ strains and the HIS4 template as described above. After 40 min incubation, scaffold isolation was carried out. NTPs were directly added to the reaction to a final concentration of 100 μM for 2 min without washing the PICs to initiate and allow only a single-round transcription. The supernatant was then removed and incubated with protein G-sepharose (Amersham) overnight at 4°C with anti-Rpb3 antibody in buffer E (20 mM HEPES (pH 8), 350 mM NaCl, 10% glycerol, 0.1% Tween 20). Beads were washed five times with buffer E and loaded onto SDS–PAGE gels together with 50 mg of input material.
