All S. cerevisiae
strains are S288C background (unless otherwise indicated), with genotypes in Table S2
. Subunits of Pol III (Rpc40 and Rpc82), TFIIIB (Bdp1 and Brf1), TFIIIC (Tfc1 and Tfc6), and Maf1 were tagged at their C termini with either 13 copies of the Myc epitope or three copies of the hemagglutinin (HA) epitope through integration at their genomic locus.
Growth Conditions and Extract Preparations
Initital growth: rich medium (1 × YP or synthetic complete medium lacking uracil with 2% glucose) grown toOD600
0.7 at 30°C (t = 0). Nutrient deprivation: cells were collected and resuspended in 0.15×YP lacking glucose or 0.15× synthetic complete media lacking uracil and glucose and grown at 30°C for the time indicated. Drug additions: 100 nM rapamycin, 0.08% (w/v) methyl methanesulfonate (MMS), and 250 μM chlorpromazine (CPZ). Extracts from crosslinked cells (for CoIP and ChIP): cells were grown in rich media to OD600
0.7, crosslinked overnight with 1% formaldehyde, washed twice in TBS, and pelleted. Pellets were lysed in ChIP lysis buffer (140 mM NaCl, 50mMHEPES, 1mMEDTA, 1%Triton X-100, and 0.1% sodium deoxycholate with protease inhibitors) by mechanical bead disruption. Lysed extracts were centrifuged, and the pellet (enriched for chromatin and membrane fractions) was retained, whereas the supernatant (enriched for cytoplasm/nucleoplasm) was discarded. The pellet was then resuspended in ChIP lysis buffer, sonicated (to release the chromatin), and recentrifuged (to remove the membrane fraction), and the supernatant was collected as the chromatin-enriched fraction. Extracts (noncrosslinked) and CoIP conditions (for both crosslinked and noncrosslinked extracts) were performed by standard methods, with details provided in the Supplemental Experimental Procedures
ChIP and qPCR
ChIP and genome-wide ChIP were performed as described previously (Roberts et al., 2003
), with modifications described in the Supplemental Experimental Procedures
. The Maf1 occupancy dataset is in Table S4
. qPCR was performed as described previously (Roberts et al., 2003
). Primers can be found in Table S3
. Occupancy in ChIP samples was determined by dividing the relative abundance of a particular region (SCR1
A–F or tRNALys[CUU]G1
) by the relative abundance of a control region (TRA1
Preparation of Extracts and Immunoprecipitations
Noncrosslinked extracts (for 2D-gel electrophoresis and radiolabeling experiments): cells were grown in selective media to an OD600 of 0.7, and after centrifugation, pellets were immediately frozen in liquid nitrogen. Pellets were thawed and washed one time in breaking buffer (50mM Tris-HCl [pH 7.5], 5mMEDTA, 12% glycerol, 0.1% Triton X-100, 250 mM NaCl, 0.5 mM DTT, protease inhibitors, and phosphatase inhibitors [2× Phosphatase Inhibitor Cocktail Set 1, Calbiochem + 0.2 mM Sodium Flouride and 0.2 mM β-Glycerophosphate]). Cells were lysed in breaking buffer by mechanical bead disruption, and after centrifugation, the supernatant was collected.
For CoIPs, extracts from crosslinked cells (600 μg) were added to 1.0 × 107 Pan Mouse IgG Dynabeads (Dynal Biotech, preincubated with BSA and either 2 μg anti-HA [12CA5] or 1.3 μg anti-Myc [9E11, Genetex]) for 4 hr at 4°C. The beads were washed four times with ChIP lysis buffer (250 mM NaCl). Samples were eluted directly into SDS sample buffer and incubated for 15 min at 90°C before running on SDS-PAGE. Antibodies for Western detection were either polyclonal anti-HA (Abcam 9110) or monoclonal anti-Myc (9E10). In , extract (800 μg) was incubated with 4.0 × 107 Pan Mouse IgG Dynabeads in low-salt IP buffer (see 2D-gel electrophoresis above) for 7 hr at 4°C and washed three times in high-salt IP buffer (see 2D-gel electrophoresis above). For determining phosphorylation status of Maf1 in association with Pol III (), 1.0 × 107 beads of HA or Myc Pan mouse IgG Dynabeads (as above) were incubated with extract (600 μg) from crosslinked YBC2077 in ChIP lysis buffer (w/250 mM NaCl) for 4 hr at 4°C. Beads were washed three times with ChIP lysis buffer (w/250 mM NaCl), one time with ChIP lysis buffer (w/375 mM NaCl), and eluted in SDS-PAGE sample buffer, immunoblotted, and probed with polyclonal anti-HA (Abcam 9110).
Two-Dimensional Gel Electrophoresis of Maf1
Whole-cell extract (800 μg) was incubated with 2.0 × 107 Pan mouse IgG Dynabeads (Dynal Biotech, preincubated with BSA and 2 μg anti-HA [12CA5]) for 4 hr at 4°C in low-salt IP buffer (50 mM Tris-HCL, 1 mM EDTA, 10% glycerol, 0.05% Tween 20, 100 mM NaCl, 0.5 mM DTT, protease inhibitors, and phosphatase inhibitors [see extract preparation]). Beads were washed three times with high-salt IP buffer (same as low-salt IP buffer except 250 mM NaCl) and resuspended in urea sample buffer + resolyte (40 mM Tris-HCL [pH 7.5], 8Murea, 4%CHAPs, 2%Bio-lyte 3–10 Ampholyte, and bromophenol blue). Samples were then subjected to either isoelectric focusing (using Readystrip IPG strips pH 3–10 [Biorad]) or SDS-PAGE.
Cultures of YBC2077 were grown to OD600 0.5 before 32P was added for 40 min, and whole-cell extracts prepared (as above). Radiolabeled extract (500 μg) was incubated with 2.0 × 107 Pan mouse IgG Dynabeads for 5 hr at 4°C. Pellets were washed four times with high-salt IP buffer (50 mM Tris-HCL, 1 mM EDTA, 10% glycerol, 0.05% Tween 20, 500 mM NaCl, 0.5 mM DTT, protease inhibitors, and phosphatase inhibitors) and incubated for an additional 4.5 hr in wash buffer. Beads were resuspended in SDS sample buffer, and the supernatant was separated on a 7.5% acrylamide gel.
Phosphatase Treatment of Maf1
Crosslinked extract (100 μg) was incubated with 1.0 × 107 Pan mouse IgG Dynabeads (Dynal Biotech, preincubated with BSA and 2 μg anti-HA [12CA5]) for 4 hr at 4°C. Beads were washed twice with ChIP lysis buffer containing 250 mM NaCl, once with ChIP lysis buffer containing 375 mM NaCl, and once with LiCl wash buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, and 1mMEDTA). Beads were washed and then equilibrated in 45 μl Lambda protein phosphatase buffer (50 mM HEPES, 100mM NaCl, 0.1mMEGTA, 2mM DTT, 0.01% Brij 35, 2 mMMnCl2, pH 7.5 at 25°C). Lambda phosphatase (800 U, NEB) was added at 30°C for 30 min, washed once with ChIP lysis buffer (375 mM NaCl), and resuspended in sample buffer for SDS-PAGE.