INTRODUCTION

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Initiation of eukaryotic mRNA synthesis in vitro requires RNA polymerase II (Pol II) and the general transcription factors (GTFs), including TBP, TFIIB, TFIIE, TFIIF, and TFIIH (reviewed in references 46 and 49). Regulation of transcription, however, requires additional cofactors to mediate the communication between DNA-binding activators or repressors, and Pol II and the GTFs. These cofactors include the TAFs associated with TBP to form TFIID (reviewed in references 17 and 54) and the mediator proteins, including the Srbps, associated with Pol II to form the "holoenzyme" (reviewed in reference 30). Recent work from several laboratories has shown that multiple forms of these complexes exist in different cell types and under different growth conditions (reviewed in reference 6). The different cofactor complexes therefore contribute to the complex regulatory patterns of eukaryotic genes.

We have reported the isolation and characterization of a novel form of Pol II in yeast distinct from the Srbp containing "holoenzyme" (51, 58). This Pol II complex contains the GTFs TFIIB and TFIIF but lacks TBP and TFIIH (51). The Gal11p-Sin4p-Rgr1p subcomplex is found in both forms of Pol II (35, 51). The products of the CDC73 and PAF1 genes are present in the novel form of Pol II but are not found in the Srbp-containing holoenzyme. Cdc73p and Paf1p are localized in the nucleus, and deletion of either gene causes pleiotropic phenotypes, including temperature sensitivity and slow growth (52). In contrast to the requirement for some of the Srbps for transcription of most yeast genes (55), the expression of only a few genes is affected by mutations in PAF1 and CDC73 (51, 52).

Why does yeast contain at least two complex initiating forms of Pol II? In this work we have begun to answer this question by identifying two additional proteins found uniquely in the Paf1p-Cdc73p complex but not in the Srbp-containing form of Pol II. These proteins, Ccr4p and Hpr1p, have both been implicated in the transcription of subsets of yeast genes (11, 63). Both factors have also been demonstrated to have genetic interactions with components of the transcription machinery (16, 38). By analyzing the phenotypes of single and multiple mutants of these Pol II-associated proteins, we have found that the complex appears to play a role both in recombination and in the expression of genes involved in cell wall biosynthesis. A feature that links these two seemingly disparate properties is a known connection to the protein kinase C-mitogen-activated protein (MAP) kinase signaling pathway (23, 25).

In yeast the protein kinase C homologue encoded by the PKC1 gene has been shown to play an essential role in maintenance of cell integrity. A mutation in PKC1 leads to cell lysis, which can be rescued by the osmotic stabilizer sorbitol (32, 47). Pkc1p is activated in response to alterations in the cell membrane caused by heat shock, hypoosmotic shock, or -factor treatment (27, 62). Activated Pkc1p initiates the sequential activation of its downstream MAP kinase cascade, including Bck1p, Mkk1p-Mkk2p, and Mpk1p (Slt2p) (reviewed in reference 33). The status of the membrane may be signaled to Pkc1p by the putative transmembrane protein Slg1p (18) through a step requiring Rho1p, a small GTP-binding protein (45). Activation of Pkc1p is critically important for cell cycle progression (34), where it plays a role in bud emergence in response to signals from Cdc28p (18, 43). Pkc1p is also sensitive to the mating pathway of yeast via direct communication with the Ste20p-activated MAP kinase cascade (5). In both bud emergence and the mating pathway, activation of Pkc1p and the MAP kinase cascade controls increased synthesis of gene products required for the newly made cell walls (25).

Pkc1p also plays a less-well-characterized role in recombination in yeast. Huang and Symington (22, 23) found that mutations in PKC1 led to increased rates of mitotic recombination but that, unlike the case for expression of the cell wall biosynthetic genes, a direct connection to the MAP kinase cascade was not evident. Our discovery of a Pol II complex containing factors that affect both the expression of cell wall biosynthetic genes and frequency of recombination therefore may begin to resolve these two very different functions of Pkc1p. Our results are consistent with the possibility that the Pol II complex containing Cdc73p, Paf1p, Ccr4p, and Hpr1p functions, at least in part, to transmit signals from the PKC1 kinase cascade to target genes involved in recombination and cell wall integrity.

	MATERIALS AND METHODS

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Strains and media. The Saccharomyces cerevisiae strains used in this study are shown in Table 1. Strains YJJ564, YJJ577, YJJ662, YJ664, YJJ665, YJJ681, YJJ691, YJJ693, YJJ832, YJJ854, YJJ875, YJJ879, YJJ898, YJJ899, YJJ932, YJJ935, YJJ952, YJJ954, YJJ956, and YJJ1027 were all derived from the homozygous diploid YJJ453 and are therefore isogenic. YJJ998 was created by mating YJJ577 with YJJ662 and then disrupting a single copy of the MPK1 gene in the diploid by using the construct described below. YJJ1027 and YJJ756 are isogenic with YJJ755. The HKY strains used for the recombination analyses are isogenic (16). Yeast strains were grown in YPD or synthetic medium prepared according to standard methods (19).

Protein-protein interaction assays. Strains containing glutathione S-transferase (GST)-tagged forms of Tfg2p and Cdc73p have been previously described (51). hpr1 strains containing the pGEST vector or a GST-Hpr1p construct were created as follows. The HPR1 coding region was amplified by PCR with primer 1 (5'-CGCGGATCCATGTCTAATACCGAGGAATTG-3') and primer 2 (5'-GCGGGATCCTTATTTCATATCTTGGGTAGATG-3'). Both primers are flanked by BamHI sites. The PCR product was digested with BamHI and ligated into pJJ560 (pGEST vector) to make an in-frame GST-HPR1 fusion under the control of a GAL promoter. The pGEST vector and the pGEST-Hpr1p construct were transformed into strain YJJ899 to create strains YJJ954 and YJJ952, respectively. The presence of the GST-Hpr1p construct, but not the vector alone, corrected the slow growth and temperature-sensitive (ts) defect of the hpr1 strain. The expression of GST-HPR1 in the hpr1 strain was confirmed by Western blot with antibody directed against Hpr1p. Transcriptionally active whole-cell extracts were prepared from yeast strains YJJ691, YJJ693, YJJ854, YJJ855, YJJ952, and YJJ954 as described previously (61). The protein concentration of the whole-cell extracts were measured by using reagents from Bio-Rad. Equal amounts of the extracts were mixed with glutathione-agarose beads in SK(20) (20 mM HEPES [pH 7.9], 20% glycerol, 10 mM MgSO₄, 10 mM EGTA, 5 mM dithiothreitol [DTT], 20 mM potassium acetate, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg of leupeptin per ml, 0.4 µg of bestatin per ml, 0.35 µg of pepstatin A per ml), incubated at 4°C for 2 to 4 h, and washed once with SK(20) and several times with SK(200) (30 mM HEPES [pH 7.9], 10% glycerol, 1 mM EDTA, 1 mM DTT, 200 mM potassium acetate, and protease inhibitors as specified above). The glutathione-agarose beads were spun down briefly and eluted with the sample buffer. The samples were resolved on a sodium dodecyl sulfate (SDS)-10% polyacrylamide gel (20). Western blot analysis was performed as described by using either alkaline phosphatase or enhanced chemiluminescence (ECL) for detection (20). The sources of the antibodies used were as follows: anti-Paf1p (52), anti-Cdc73p (51), anti-Hpr1p- from M. Christman, anti-Ccr4p (42), anti-Gal11p- from T. Fukasawa, anti-Srb5p- from R. Young, and anti-TFIIS and anti-TBP (57).

Differential display and yeast mRNA analysis. Total yeast RNA was isolated as described previously (13). Differential display analysis has been described (51). RNA samples for Northern blots were quantitated by measuring the optical density at 260 nm, equal amounts of RNA were run on 1% agarose-formaldehyde gels, and RNA blots were prepared according to standard methods (50). [-³²P]dATP-labeled probes were prepared by random priming (50). An 18S rRNA oligonucleotide probe was labeled with T4 kinase and [-³²P]ATP (50). Northern blots were quantitated by PhosphorImager analysis.

Double deletion and tetrad analysis. Appropriate deletion strains were crossed to obtain the desired diploid strains, which were sporulated, and at least 30 tetrads for each diploid were dissected (19). The genotypes of the individual spores were determined by analysis of the different markers used to replace the deleted genes. In some cases, PCR was used to confirm the genotype of the spores.

Construction of deletion strains. SRB5 coding and flanking regions were amplified by using primer 1 (5'-TGCAGCAGCTAAACCTCCAC-3') and primer 2 (5'-GACGATGACGAAGAGCTAC-3'). The PCR product was ligated into pGEM-T (Promega) vector. Primer 3 (5'-ACGCAAGCTTTTCTTCTTAATATGGAATAC-3') and primer 4 (5'-GACGAAGCTTATAATCATTGGCACCTGG-3') were used to amplify the resulting plasmid. The PCR product was then cut with HindIII and ligated with the 1.2-kb HindIII URA3 fragment from YEp24 (4) to give pJJ995. pJJ995 was cut with HindIII, blunt ended with Klenow, and ligated with the BamHI/XhoI-cut and Klenow-treated 1.4-kb HIS3 fragment from YIp1 (53) to give pJJ1072. pJJ995 or pJJ1072 was cut with SpeI/SacII and used to transform YJJ453. The MPK1 deletion construct was made as follows. MPK1 coding and flanking regions were amplified with primer 1 (5'-ATGGCTGATAAGATAGAGAGG-3') and primer 2 (5'-AGGAATTCAAGAGGCGATAAC-3'). The PCR product was ligated into the PCRII (Invitrogen) vector. The resulting plasmid was partially digested with HindIII, and the 4.2-kb fragment was ligated with the 1.2-kb HindIII URA3 fragment from YEp24 (4) to give pJJ1143. pJJ1143 was cut with EcoRI and used to transform YJJ453. The CCR4 deletion construct was as described earlier (42). The SIN4 deletion construct was derived from M1381 containing sin4::LEU2 (from D. Stillman) which is similar to the sin4::TRP1 disruption described by Jiang and Stillman (26). The HPR1 deletion construct hpr1::HIS3 was as described earlier (2). The deletion constructs described above were transformed into diploid strain YJJ453. The paf1::TRP1 disruptor was made as follows. Primer P1 (5'-CTTAGCACAACTGAATTCGAAAGG-3') and primer P4 (5'-ATACGAATGATGTTAATGGAGACTCCAGGATTGTCGACT-3') were used to PCR amplify the paf1::HIS3 construct from genomic DNA (YJJ664). The PCR product was subcloned into the pGEMT vector (Promega) to give pJJ902. pJJ902 was cut with XhoI/BamHI to release the 1.2-kb HIS3 fragment. The vector fragment was gel purified and ligated with the 900-bp SalI/BglII TRP1 fragment, which resulted in pJJ904. pJJ904 was linearized with SpeI/SacII and used to transform yeast strain YJJ755 to obtain paf1 strain YJJ756. The pkc1::LEU2 disruptor was made as follows. The PKC1 coding and flanking sequences were amplified from yeast genomic DNA (YJJ755) by PCR with primer 1 (5'-AACTGCAGCATGAGTTTTTCACAATTGTAG-3') and primer 2 (5'-AACTGCAGTCATGGCATGACCTTTTCTTC-3').

Enzymatic assays. CYC1 and FKS1 promoter-lacZ fusion reporter plasmids used in -galactosidase assays were as previously described (25, 28). Yeast cells transformed with the plasmids (19) were grown in Ura-Casamino Acid media supplemented with 4% glucose. Extract preparation and -galactosidase assays were performed as described elsewhere (44). Mpk1p kinase assays with hemagglutin (HA)-tagged Mpk1p to phosphorylate MBP were as described by Zarzov et al. (62).

Determination of recombination rates. Recombination rates were calculated according to the median method of Lea and Coulson (31) as described previously (1).

	RESULTS

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Ccr4p and Hpr1p are in the Paf1p-Cdc73p-Pol II complex. Paf1p and Cdc73p were originally identified as proteins associated with a transcriptionally active form of Pol II immobilized by an antibody directed against the nonphosphorylated form of the C-terminal domain (CTD [58]). The proteins bound to Pol II and eluted with moderate salt included TFIIB, the subunits of TFIIF, TFIIS, Paf1p, Cdc73p, Gal11p, and Sin4p, as well as approximately 13 other polypeptides, but did not include TBP, TFIIH, the Srbps or the Swip-Snfp factors (58). To confirm that these Pol II-associated proteins defined a form of Pol II distinct from the Srbp-containing holoenzyme, we created strains bearing GST-tagged forms of Cdc73p and Paf1p functionally replacing the CDC73 and PAF1 genes in yeast cells and analyzed the composition of the complexes isolated by using glutathione-agarose chromatography (51). These experiments confirmed that Cdc73p and Paf1p are found in a distinct complex with Pol II, TFIIB, TFIIF, Gal11p, and Sin4p. Elongation factor TFIIS apparently defines at least one additional form of Pol II, since it was not in any of the GST-tagged complexes nor is it present in the Srbp-containing holoenzyme (51).

View larger version (43K): [in this window] [in a new window]   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 tfg2 mutant complemented with GST-Tfg2p (YJJ854); lanes 5 and 6 (labeled cdc73-GST) are from cdc73 (YJJ665) mutant cells transformed with the GST vector; and lanes 7 and 8 (labeled GST-CDC73) are from the cdc73 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 hpr1-GST) are from an hpr1 strain transformed with the GST vector alone (YJJ954); lanes 3 and 4 (labeled GST-HPR1) are from an hpr1 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; -70, 10 µl of the salt-eluted fraction from a control column containing antibody directed against the 70 subunit of E. coli RNA polymerase; -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.

View larger version (122K): [in this window] [in a new window]   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), cdc73 (YJJ665), and paf1 (YJJ664) cells, respectively, transformed with the GST vector alone. Lanes 3 and 5 show cdc73 and paf1 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.

Identification of transcripts affected by mutations in Paf1p-Cdc73p-Pol II complex components. Mutations in PAF1 and CDC73 affect the transcript abundance of only a very few yeast genes (51, 52). In general, mutation of PAF1 affects the expression of more transcripts than mutation of CDC73, a finding consistent with its more severe phenotype. For example, we have previously shown that inducible GAL gene expression measured in the chromosomal GAL7+10 genes and in a GAL promoter-reporter construct is reduced five- to eightfold in paf1 but is relatively unaffected by cdc73 (52). We have also analyzed the inducible expression of the GAL1,7+10 genes in isogenic ccr4 and hpr1 strains. We found that GAL gene induction in a ccr4 strain was actually enhanced in both rate and extent, while there was little or no effect on GAL gene expression in an hpr1 strain (data not shown). Therefore, although these proteins are clearly found together in a complex, their effects on gene expression are not equivalent.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   Transcripts identified by differential display are differentially expressed in isogenic paf1, cdc73, gal11, srb5, ccr4, and hpr1 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 were paf1-YJJ664, cdc73-YJJ665, gal11-YJJ564, srb5-YJJ875, ccr4-YJJ879, and hpr1-YJJ898.

View this table: [in this window] [in a new window]   TABLE 2.   Expression of promoter-reporter constructs is reduced in the paf1 straina

Genetic interactions between PAF1, CDC73, CCR4, and HPR1. We have previously reported that a paf1 cdc73 double mutant is viable and does not show an enhanced phenotype (51), while a paf1 gal11 double mutant demonstrates a dramatically enhanced slow-growth phenotype (52). These data are consistent with the idea that Paf1p and Gal11p have at least partially overlapping essential functions in the yeast cell, with Paf1p and Cdc73p participating in the same pathway such that a double mutant is no more deleterious than either single mutation. To determine the genetic interactions between PAF1, CDC73, and the newly identified components of the Pol II complex, CCR4 and HPR1, we created double mutants in an isogenic background as described in Materials and Methods. Many of the double-mutant analyses were repeated in other genetic backgrounds with identical results. We also evaluated the effects of combining mutations in these genes with mutations in other holoenzyme components, including gal11, sin4, and srb5. Based on the results of 30 or more tetrads for each doubly mutant diploid, we found extensive evidence for genetic interactions, as summarized in Fig. 4. We found that combining a paf1 mutation with either ccr4 or hpr1 results in lethality. Deletion of CCR4 is also lethal in combination with cdc73, gal11, or hpr1 and has an enhanced phenotype with sin4. In each case the nonviable spores did germinate and formed microcolonies. The combination of cdc73 and hpr1 results in an enhanced ts phenotype in which the permissive temperature is reduced from 30 to 22°C. These results, like our earlier analysis of the paf1gal11 double mutant (51), support the idea that the components of this novel Pol II complex have redundant essential functions for the yeast cell.

View larger version (32K): [in this window] [in a new window]   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 were paf1-YJJ664 or -YJJ577; cdc73-YJJ665 or YJJ681; gal11-YJJ564; sin4-YJJ832 srb5-YJJ956, -YJJ935, or -YJJ875; ccr4-YJJ932 or -YJJ879 and hpr1-YJJ898 or -YJJ899.

The Paf1p-Cdc73p-Pol II complex is involved in recombination. The differential display and genetic analyses described above suggested that factors within the Paf1p-Cdc73p-Pol II complex have some similar functions. Aguilera and Klein (1) reported that the hpr1 mutation leads to increased levels of recombination between direct repeats. We therefore asked if mutations in the other factors in the Paf1p-Cdc73p-Pol II complex demonstrated a similar phenotype. As shown in Table 3, the recombination rates in the paf1 and cdc73 strains increased 82- and 45-fold, respectively, relative to the wild type, while a 700-fold increase was observed in the hpr1 strain. In contrast, recombination rates were essentially unchanged relative to wild type in ccr4, sin4, and srb5 strains. This result strongly supports the idea that defects in some components of a Pol II complex, specifically the Paf1p-Cdc73p-Pol II complex, lead to elevated levels of recombination. Whether the complex is directly involved in recombination or the formation of a recombinogenic substrate, or indirectly affecting the expression of genes required for this process is discussed further below.

Cdc73p, Paf1p, and Ccr4p are required for the integrity of the cell wall. Both paf1 and ccr4 show enlarged cell morphology and use glycerol as a carbon source very poorly (52, 10). In addition, ccr4 is hypersensitive to staurosporine and to 8 mM caffeine and 0.04% SDS (21, 37, 38), an indication of weakened cell walls (8, 39). We therefore tested the possibility that other components of the Paf1p-Cdc73p-Pol II complex might also have cell wall defects. As shown in Fig. 5, both paf1 and ccr4 are hypersensitive to growth on 8 mM caffeine (Fig. 5B) and somewhat sensitive to growth on 20 µg of calcofluor per ml (Fig. 5D), another assay for cell wall integrity (39, 48). These mutant strains and the gal11 strain are inhibited by growth on 0.02% SDS (Fig. 5C). A plasmid expressing PAF1 can correct the caffeine and calcofluor sensitivity phenotypes of paf1, confirming that the phenotypes are caused by deletion of the PAF1 gene (data not shown). In contrast, isogenic cdc73, srb5, hpr1, and sin4 strains are no more sensitive to these cell wall-damaging agents than is the wild-type strain (Fig. 5).

View larger version (77K): [in this window] [in a new window]   FIG. 5.   Mutations in PAF1, CCR4, and GAL11 lead to increased sensitivity to cell wall-damaging agents. Isogenic wild type (WT; YJJ662) and paf1 (YJJ664), cdc73 (YJJ665), gal11 (YJJ564), srb5 (YJJ875), ccr4 (YJJ879), sin4 (YJJ832), and hpr1 (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.

View larger version (59K): [in this window] [in a new window]   FIG. 6.   The ts phenotype of paf1, cdc73, ccr4, and gal11 can be corrected by the cell wall-stabilizing agent sorbitol. (A) Isogenic wild type (WT; YJJ662), paf1 (YJJ664), cdc73 (YJJ665), gal11 (YJJ564), srb5 (YJJ875), ccr4 (YJJ879), sin4 (YJJ832), and hpr1 (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 paf1 (YJJ664) strains were grown on YPD or YPD plus 1 M sorbitol at 35.5°C for 2.5 days.

Expression of cell wall biosynthetic genes is affected by paf1, cdc73, and ccr4. The two phenotypes described above, i.e., increases in recombination between direct repeats and defects in cell wall integrity, have both been observed for mutations in yeast protein kinase C (PKC1) (23, 47). Although the direct connection between recombination and PKC1 is not yet clear, Igual et al. (25) have provided a partial explanation for the cell wall defects by measuring a reduction in expression of several key genes in cell wall biosynthesis (FKS1, MNN1, GAS1, KRE6, and VAN2) in pkc1 and mpk1 mutant strains. We therefore analyzed the effects of mutations in the Paf1p-Cdc73p-Pol II complex on the expression of the FKS1, MNN1, GAS1, KRE6, and VAN2 genes as shown in Fig. 7. A representative example of some of the RNA analyses are shown in Fig. 7A, and a quantitation of three independent sets of samples are shown in Fig. 7B. We found that the paf1 mutation causes a two- to threefold decrease in the expression of several of these genes at the permissive temperature, and a four- to fivefold reduction in FKS1 expression at 38°C. For comparison, Igual et al. (25) reported that mutations in PKC1 and in MPK1 (SLT2), the terminal MAP kinase in the PKC1 signaling cascade, reduced FKS1 mRNA abundance from 1.5- to 2-fold at 30°C and from 3- to 4-fold at 38°C. In addition, they found that MNN1 and GAS1 expression was reduced two- to threefold, while there was little affect on the abundance of the KRE6 and VAN2 mRNAs (25).

View larger version (28K): [in this window] [in a new window]   FIG. 7.   Expression of genes involved in cell wall biosynthesis is reduced in paf1 ccr4 and gal11 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, paf1-YJJ664, cdc73-YJJ665, gal11-YJJ564, srb5-YJJ875, ccr4-YJJ879, and hpr1-YJJ898.

The Paf1p-Cdc73p-Pol II complex functions in the Pkc1p-Mpk1p MAP kinase cascade. If the Paf1p-Cdc73p-Pol II complex functions in the same pathway as the Pkc1p-MAP kinase cascade, then combining a mutation in PKC1 or MPK1 with a PAF1 mutation should not have a more deleterious effect on cell wall integrity than either single mutation. The phenotype of mutations in MPK1 are less severe and more variable in different strains than is the phenotype of a mutation in PKC1 (43). We therefore created paf1 mpk1 double mutants in two different genetic backgrounds which varied in the extent of the effect of the mpk1 mutation. Spores dissected from both mpk1-MPK1 paf1-PAF1 heterozygous diploids were all viable (Fig. 8A). The double-mutant spores were slow growing, ts and caffeine sensitive, and indistinguishable from the paf1 parent. We also created paf1 pkc1 double mutants and again found that all of the doubly mutant spores were viable and dependent on sorbitol like the pkc1 parent (data not shown). The paf1 pkc1 double mutant did grow somewhat more slowly than either of the single mutations. However, when we analyzed the abundance of FKS1 mRNA in the paf1 mpk1 and paf1 pkc1 double mutants we found no additional reduction in expression relative to the single mutants (data not shown). We conclude from these results that the similar effects of the PAF1 gene and the PKC1 and MPK1 genes on cell wall biosynthesis are due to their participation in the same regulatory pathway.

View larger version (35K): [in this window] [in a new window]   FIG. 8.   Interactions between PAF1 and MPK1. (A) A strain heterozygous for paf1 and mpk1 (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, mpk1::URA3; p, paf1::HIS3; mp, mpk1paf1. (B) Cell extracts were prepared from wild type (WT; YJJ755) and paf1 (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.

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

A large and growing collection of proteins has been identified as mediators or coactivators critical for communicating signals from transcriptional activators and repressors to RNA Pol II (reviewed in reference 6). In this work we have extended the description of a unique collection of proteins associated with Pol II, each of which appears to function as a transcriptional mediator. The first components of this complex to be identified, Paf1p and Cdc73p, are not encoded by essential genes but are required for the normal expression of a subset of yeast genes (51, 52). In this work we have shown that Ccr4p and Hpr1p, also encoded by nonessential genes, are also components of this Pol II complex, where they play a role in the expression of subsets of yeast genes. Although mutations in PAF1, CDC73, HPR1, and CCR4 lead to complex changes in expression patterns and, in some cases, to very different phenotypes, we have established that many of the functions of the Pol II complex are consistent with a specific role in the protein kinase C signaling cascade. In particular we have found that Paf1p, Ccr4p, and possibly Cdc73p play a role in the expression of genes required for cell wall biosynthesis and that Paf1p, Cdc73p, and Hpr1p are required for normal levels of recombination between direct repeats. As outlined in the introduction, similar effects on both the expression of cell wall biosynthetic genes and on recombination have been associated with defects in PKC1 (see Fig. 9).

Paf1p, Cdc73p, Hpr1p, and Ccr4p are found together in a complex with Pol II. We have shown that Hpr1p and Ccr4p are present in the previously described Paf1p-Cdc73p-Pol II complex. Two very different isolation procedures, affinity isolation of a transcriptionally active form of RNA Pol II and isolation of GST-tagged forms of Cdc73p, Paf1p, Hpr1p, and Tfg2p have established that these four proteins exist in a complex with RNA Pol II, TFIIB, TFIIF, and the Gal11p-Rgr1p-Sin4p subcomplex. None of these proteins is present in the Srbp-containing form of Pol II (38, 51, 63), which further supports our earlier evidence that the complex is biochemically distinct from the mediator-containing "holoenzyme." We cannot currently rule out the possibility that more than one complex containing subsets of these proteins exists in yeast cells, but the fact that Hpr1p and Ccr4p colocalize with tagged forms of both Cdc73p and Paf1p, and the fact that Ccr4p, Paf1p, and Cdc73p are all found in a tagged Hpr1p complex is consistent with this being a single complex. Does this complex, like the Srbp-containing form of Pol II (29), have homologues in other eukaryotes? Currently, there are no examples of homologues of Paf1p, Cdc73p, or Hpr1p, although homologues of Ccr4p can be found in the database. Since the Paf1p-Cdc73p form of Pol II is present in low amounts in yeast cells (51), the failure to have observed homologues in other systems may be due to low abundance of the gene products or a restricted distribution of the factors.

Phenotypes of paf1, cdc73, hpr1, and ccr4 mutations are consistent with a role in Pkc1p signaling. Although the phenotypes of mutations in the different Pol II-associated factors are not identical, there are some shared patterns, including the facts that mutations in PAF1 and CCR4 cause sensitivity to caffeine, calcofluor, and SDS (Fig. 5); the ts phenotype of mutations in CDC73 and CCR4, and to a lesser extent, PAF1 can be rescued by sorbitol (Fig. 6); and mutations in HPR1, PAF1, and CDC73 all result in elevated rates of recombination (Table 3).

Connections between transcription and recombination. To establish that the effects of the mutations in the Pol II-associated factors were at the level of transcriptional initiation, we used promoter-reporter constructs to confirm the results of the RNA analysis described above. Although the results of the -galactosidase reporter assays agreed with the mRNA analyses, the effects were more dramatic in the reporter assays. For example a PAF1 mutation reduces the level of FKS1 mRNA 2.5-fold but reduces the level of expression from the FKS1 promoter construct nearly 8-fold (Fig. 7 and Table 2). We saw similar effects with CYC1 mRNA and a CYC1 reporter construct with 3- to 4-fold and 12-fold reductions, respectively, in a paf1 background (Fig. 3 and Table 2). This same phenomenon has been observed by Fan and Klein (14) and Chavez and Aguilera (7) for expression of GAL10 and GAL1 mRNAs versus expression from promoter-fusion constructs in an HPR1 mutant background. Chavez and Aguilera (7) have shown that the discrepancy is due to an elongation pause site in the lacZ coding region causing reduced abundance of full-length RNA in hpr1 cells. Does the fact that we see a similar discrepancy for paf1 indicate that these genes play a direct role in transcriptional elongation? This seems unlikely since we have shown that unlike GAL1 expression in hpr1, the level of GAL1,7+10, CYC1, and FKS1 mRNAs (plus many other mRNAs shown in Fig. 3 and 7) are significantly reduced in paf1 and, in some cases, the ccr4 and cdc73 backgrounds. It is probable that the additional effects that we see with the reporter constructs are due to secondary effects on elongation through the lacZ gene.

The Paf1p-Cdc73p-Pol II complex is downstream of the Pkc1p-MAP kinase cascade. As described above, many of the phenotypes of mutations in the PKC1-MPK1 pathway are mimicked by mutations in PAF1, CDC73, CCR4, and HPR1. The observation that paf1 mpk1 and paf1 pkc1 double mutants do not demonstrate an enhanced phenotype (Fig. 8 and data not shown), strongly supports the theory that all of these factors function in the same pathway. Our results do not rule out the possibility that the Pol II complex is integrating inputs from more than one signaling pathway. The fact that many double mutants of factors within the complex (paf1 ccr4, paf1 hpr1, cdc73 ccr4, and ccr4 hpr1) are synthetically lethal and cannot be rescued by sorbitol is consistent with this view. Our results are therefore in agreement with the conclusions of Hata et al. (21), who determined that Ccr4p might be functioning in both the Pkc1p and an independent Caf1p(Pop2p) pathway. The existence of distinct complexes and pathways is also consistent with the fact that Caf1p is not present in the Paf1p-Cdc73p-Pol II complex.

View larger version (24K): [in this window] [in a new window]   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.