Article Figures & Data
Figures
- Fig. 1.
Hpr1p and Ccr4p are present in the Paf1p-Cdc73p-Pol II complex. Proteins separated on SDS polyacrylamide gels were transferred and probed with antibodies directed against Hpr1p and Ccr4p as indicated. ECL was used for antibody detection. (A) Transcription-competent whole-cell extracts (WCE) were isolated and used as a source to purify GST-tagged Tfg2p, Cdc73p, and associated proteins by glutathione agarose chromatography as described in Materials and Methods. Lanes 1, 3, 5, and 7 (labeled IP) contain the input WCE from the indicated strains; lanes 2, 4, 6, and 8 (labeled B) contain the proteins bound to the glutathione agarose beads. Lanes 1 and 2 (labeled WT-GST) are from wild-type (YJJ662) cells transformed with the GST vector alone; lanes 3 and 4 (labeled GST-TFG2) are from the tfg2Δ mutant complemented with GST-Tfg2p (YJJ854); lanes 5 and 6 (labeled cdc73Δ-GST) are fromcdc73Δ (YJJ665) mutant cells transformed with the GST vector; and lanes 7 and 8 (labeled GST-CDC73) are from thecdc73Δ mutant complemented by GST-Cdc73p (YJJ691). (B) Transcription-competent WCE were isolated and used as a source to purify GST-tagged Hpr1p and associated proteins by glutathione agarose chromatography as described in Materials and Methods. Lanes labeled IP and B are as described in panel A. Lanes 1 and 2 (labeled hpr1Δ-GST) are from an hpr1Δ strain transformed with the GST vector alone (YJJ954); lanes 3 and 4 (labeled GST-HPR1) are from anhpr1Δ strain transformed with GST-Hpr1p (YJJ952). (C) Fractions from antibody affinity chromatography performed as described in Wade et al. (58). Lanes: WCE, 40 μg of protein from a transcription competent WCE; α-ς70, 10 μl of the salt-eluted fraction from a control column containing antibody directed against the ς70 subunit of E. coli RNA polymerase; α-CTD, 10 μl of the salt-eluted fraction from a column containing antibody directed against the C-terminal domain of the largest subunit of RNA Pol II.
- Fig. 2.
A mutation in the CDC73 gene affects the abundance of Ccr4p, Hpr1p, Gal11p, and Paf1p. The abundance of the indicated proteins was analyzed as described in Materials and Methods in different transcription-competent WCEs. Antibodies were detected with alkaline phosphatase. Lanes 1, 2, and 4 show WCEs from wild-type (WT; YJJ662), cdc73Δ (YJJ665), and paf1Δ (YJJ664) cells, respectively, transformed with the GST vector alone. Lanes 3 and 5 show cdc73Δ and paf1Δ mutant strains complemented by GST-Cdc73p (YJJ691) and GST-Paf1p (YJJ676), respectively. The arrowhead above Paf1p points to the position of the GST-Paf1p protein seen in lane 5. Proteins absent from the Paf1p-Cdc73p-Pol II complex, including Srb5p, TFIIS, and TBP, are used as loading controls.
- Fig. 3.
Transcripts identified by differential display are differentially expressed in isogenic paf1Δ,cdc73Δ, gal11Δ, srb5Δ,ccr4Δ, and hpr1Δ mutant strains. Differential display was performed as described in Materials and Methods. DNA encoding the differentially expressed transcripts was cloned and sequenced to identify the yeast gene. RNA was isolated from the indicated isogenic strains and probed for transcripts from each gene as described in Materials and Methods. Abundance was determined with a PhosphorImager and was normalized to the signal for 18S rRNA. The data is presented relative to a transcript abundance in wild type set as 1, which is shown as a dashed line in each panel. The results shown represent the averages and standard deviations from six to nine separate RNA isolations. The yeast strains used for RNA isolation werepaf1Δ-YJJ664, cdc73Δ-YJJ665,gal11Δ-YJJ564, srb5Δ-YJJ875,ccr4Δ-YJJ879, and hpr1Δ-YJJ898.
- Fig. 4.
Synthetic genetic interactions between pairwise combinations of factors in RNA Pol II complexes. Tetrad analysis of heterozygous diploids obtained by sporulating crosses between isogenic single deletion mutants and tetrad dissection. At least 30 tetrads were dissected for each diploid analyzed. All of the single deletion mutants are ts at 38°C. The growth phenotypes of the double-deletion mutants are shown in the figure, with shaded areas highlighting significant synthetic interactions (solid box, synthetic lethality; gray box, synthetic enhancement). The genotypes of inviable spores were deduced by the markers used to delete the genes. “Slow growth” means that the double mutants grew significantly more slowly than either of the parents. The “ts at 30°” means that the permissive temperature for the double mutant is reduced to 22°C. The “ts” in an unshaded box indicates that the phenotype of the double mutant was not significantly worse than either of the parent strains. The asterisks refer to previously published results (51, 52) included for completeness. The strains used in the crosses werepaf1Δ-YJJ664 or -YJJ577; cdc73Δ-YJJ665 or YJJ681; gal11Δ-YJJ564; sin4Δ-YJJ832srb5Δ-YJJ956, -YJJ935, or -YJJ875;ccr4Δ-YJJ932 or -YJJ879 and hpr1Δ-YJJ898 or -YJJ899.
- Fig. 5.
Mutations in PAF1, CCR4, andGAL11 lead to increased sensitivity to cell wall-damaging agents. Isogenic wild type (WT; YJJ662) and paf1Δ (YJJ664), cdc73Δ (YJJ665), gal11Δ (YJJ564),srb5Δ (YJJ875), ccr4Δ (YJJ879),sin4Δ (YJJ832), and hpr1Δ (YJJ898) were grown on YPD or YPD plus the indicated additions. The cells were allowed to grow at 30°C for 3 to 4 days.
- Fig. 6.
The ts phenotype of paf1Δ,cdc73Δ, ccr4Δ, and gal11Δ can be corrected by the cell wall-stabilizing agent sorbitol. (A) Isogenic wild type (WT; YJJ662), paf1Δ (YJJ664),cdc73Δ (YJJ665), gal11Δ (YJJ564),srb5Δ (YJJ875), ccr4Δ (YJJ879),sin4Δ (YJJ832), and hpr1Δ (YJJ898) strains were grown on YPD or YPD plus 1 M sorbitol at 38°C for 4 days. (B) Isogenic wild-type (WT; YJJ662) and paf1Δ (YJJ664) strains were grown on YPD or YPD plus 1 M sorbitol at 35.5°C for 2.5 days.
- Fig. 7.
Expression of genes involved in cell wall biosynthesis is reduced in paf1Δ ccr4Δ and gal11Δ mutants. (A) Total yeast RNA was prepared from isogenic mutant strains and probed for the indicated genes as described in Materials and Methods. (B) The results shown are based on three or more independently isolated sets of RNA. The RNA abundance was normalized to 18S rRNA, and the wild-type value is set as 1, which is shown as a dashed line in each panel. The RNA in the panel labeled FKS1 at 38°C was isolated from cells shifted from 30 to 38°C and incubated for 5 h. The yeast strains used for RNA isolation were wild type-YJJ662,paf1Δ-YJJ664, cdc73Δ-YJJ665,gal11Δ-YJJ564, srb5Δ-YJJ875,ccr4Δ-YJJ879, and hpr1Δ-YJJ898.
- Fig. 8.
Interactions between PAF1 andMPK1. (A) A strain heterozygous for paf1Δ andmpk1Δ (YJJ998) was sporulated, and tetrads were dissected. The figure shows 6 of 30 tetrads, all of which showed similar results. The genotype of the spores was determined from the markers associated with the deletions. wt, Wild type; m,mpk1Δ::URA3; p, paf1Δ::HIS3; mp,mpk1Δpaf1Δ. (B) Cell extracts were prepared from wild type (WT; YJJ755) and paf1Δ (YJJ756) strains containing a 3HA-tagged form of Mpk1p and grown at the indicated temperatures. The tagged Mpk1p was isolated and used to phosphorylate the MAP kinase substrate MBP as described by Zarzov et al. (62). The data represent the phosphorylation of MBP normalized to the amount of HA-tagged Mpk1p in each extract.
- Fig. 9.
A model for the interactions between the Paf1p-Cdc73p-Pol II transcription complex and the Pkc1p-Mpk1p protein kinase cascade. An explanation of the model is provided in the text.
Tables
- Table 1.
Yeast strains used in this study
Strain Genotype Reference or source YJJ453 MAT a/αleu2Δ1/leu2Δ1 his3Δ200/his3Δ200 ura3-52/ura3-52 56 YJJ564 MAT a leu2Δ1 his3Δ200 ura3-52 gal11Δ::LEU2 52 YJJ577 MATα leu2Δ1 his3Δ200 ura3-52 paf1Δ::HIS3 52 YJJ662 MAT a leu2Δ1 his3Δ200 ura3-52 52 YJJ664 MAT a leu2Δ1 his3Δ200 ura3-52 paf1Δ::HIS3 52 YJJ665 MAT a leu2Δ1 his3Δ200 ura3-52 cdc73Δ::HIS3 52 YJJ681 MATα leu2Δ1 his3Δ200 ura3-52 cdc73Δ::HIS3 X. Shi YJJ691 MAT a leu2Δ1 his3Δ200 ura3-52 cdc73Δ::HIS3(pEGST-CDC73) 52 YJJ693 MAT a leu2Δ1 his3Δ200 ura3-52 cdc73Δ::HIS3(pEGST) 52 YJJ755 MAT a bar1 his6 his7 leu2 ura3 pep4 prb1 trp1 R. Sclafani YJJ756 MAT a bar1 his6 his7 leu2 ura3 pep4 prb1 trp1 paf1Δ::TRP1 This work YJJ832 MAT a leu2Δ1 his3Δ200 ura3-52 sin4::LEU2 This work YJJ854 MAT a leu2Δ1 his3Δ200 ura3-52 trg2Δ::HIS3(pEGST-TFG2) 52 YJJ855 MAT a leu2Δ1 his3Δ200 ura3-52(pEGST) 52 YJJ875 MAT a leu2Δ1 his3Δ200 ura3-52 srb5Δ::URA3 This work YJJ879 MAT a leu2Δ1 his3Δ200 ura3-52 ccr4Δ::URA3 This work YJJ898 MAT a leu2Δ1 his3Δ200 ura3-52 hpr1Δ::HIS3 This work YJJ899 MATα leu2Δ1 his3Δ200 ura3-52 hpr1Δ::HIS3 This work YJJ932 MATα leu2Δ1 his3Δ200 ura3-52 ccr4Δ::URA3 This work YJJ935 MATα leu2Δ1 his3Δ200 ura3-52 srb5Δ::URA3 This work YJJ956 MAT a leu2Δ1 his3Δ200 ura3-52 srb5Δ::HIS3 This work YJJ952 MATα leu2Δ1 his3Δ200 ura3-52 hpr1Δ::HIS3(pEGST-HPR1) This work YJJ954 MATα leu2Δ1 his3Δ200 ura3-52 hpr1Δ::HIS3(pEGST) This work YJJ998 MAT a /MATα leu2Δ1/leu2Δ1 his3Δ200/his3Δ200 ura3-52/ura3-52 PAF1/paf1Δ::HIS3 MPK1/mpk1Δ::URA3 This work YJJ1027 MATα his3Δ1 leu2 trp1 ura3 can1 cyh2 gal1 R. Sclafani YJJ1036 MAT a /MATα bar1 his3Δ1 his6 his7 leu2/leu2 ura3/ura3 pep4 prb1 trp1/trp1 can1 cyh2 gal1 This work HKY870-12A MAT a leu2-k::ADE2-URA3::leu2-k ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100 15 HFY998-2C MAT a leu2-k::ADE2-URA3::leu2-k ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100 hpr1::HIS3 15 HFY2074 MAT a leu2-k::ADE2-URA3::leu2-k ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100 paf1::HIS3 15 HFY2059-1A MAT a leu2-k::ADE2-URA3::leu2-k ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100 cdc73::HIS3 15 HFY2085 MAT a leu2-k::ADE2-URA3::leu2-k ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100 ccr4::HIS3 15 HFY2162 MATα leu2-k::ADE2-URA3::leu2-k ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100 sin4::TRP1 15 HFY2069 MAT a leu2-k::ADE2-URA3::leu2-k ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100 srb5::HIS3 15 - Table 2.
Expression of promoter-reporter constructs is reduced in the paf1Δ straina
Strain β-Galactosidase activity (U/mg) with promoter: CYC1 FKS1 Wild type 24,000 ± 1,500 20,000 ± 3,200 paf1Δ 1,900 ± 370 2,600 ± 150 ↵a Extracts from wild-type (YJJ662) andpaf1Δ mutant (YJJ664) strains bearing the pF712-380pFKS1-lacZ reporter plasmid (25) or the AJ-1CYC1-lacZ reporter plasmid (28) were prepared and assayed as described in Materials and Methods. β-Galactosidase activity (± the standard error of the mean) is expressed in units per milligram of protein, where 1 U is defined as the A420 × 103 per minute. Values are derived from replicate assays of at least three different transformants.
- Table 3.
Mutations in HPR1, PAF1, andCDC73 lead to elevated rates of recombination between direct repeats
Genotypea Recombination rate (10−6)b Fold increase Wild type 3.8 ± 1.8 1 hpr1Δ 2,700 ± 1,400 700 paf1Δ 320 ± 50 82 cdc73Δ 170 ± 40 45 ccr4Δ 4.1 ± 1.9 1 sin4Δ 3.0 ± 1.7 1 srb5Δ 9.2 ± 4.8 2 ↵a Strains used in this table are wild type (HKY870-12A), hpr1Δ (HFY988-2C), paf1Δ (HFY2074), cdc73Δ (HFY2059-1A), ccr4Δ (HFY2085), sin4Δ (HFY2162), and srb5Δ (HFY2069).
↵b Rates were calculated as described in Materials and Methods from 5-FOA resistance frequencies of strains carrying the duplicationleu2-k::ADE2-URA3::leu2-k. Each rate was calculated from three independent fluctuation tests on three strains of the same genotype and is expressed as the mean rate ± the standard deviation of the three determinations.
