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J. Biol. Chem., Vol. 276, Issue 33, 30641-30647, August 17, 2001

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Rpb4, a Non-essential Subunit of Core RNA Polymerase II of Saccharomyces cerevisiae Is Important for Activated Transcription of a Subset of Genes^*

Beena Pillai, Vinaya Sampath, Nimisha Sharma, and Parag Sadhale

From the Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India 560 012

Received for publication, December 5, 2000, and in revised form, May 26, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A major role in the regulation of eukaryotic protein-coding genes is played by the gene-specific transcriptional regulators, which recruit the RNA polymerase II holoenzyme to the specific promoter. Several components of the mediator complex within the holoenzyme also have been shown to affect activation of different subsets of genes. Only recently has it been suggested that besides the largest subunit of RNA polymerase II, smaller subunits like Rpb3 and Rpb5 may have regulatory roles in expression of specific sets of genes. We report here, the role of Rpb4, a non-essential subunit of core RNA polymerase II, in activation of a subset of genes in Saccharomyces cerevisiae. We have shown below that whereas constitutive transcription is largely unaffected, activation from various promoters tested is severely compromised in the absence of RPB4. This activation defect can be rescued by the overexpression of cognate activators. We have localized the region of Rpb4 involved in activation to the C-terminal 24 amino acids. We have also shown here that transcriptional activation by artificial recruitment of the TATA-binding protein (TBP) to the promoter is also defective in the absence of RPB4. Surprisingly, the overexpression of RPB7 (the interacting partner of Rpb4) does not rescue the activation defect of all the promoters tested, although it rescues the activation defect of the heat shock element-containing promoter and the temperature sensitivity associated with RPB4 deletion. Overall, our results indicate that Rpb4 and Rpb7 play independent roles in transcriptional regulation of genes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transcriptional regulation at the initiation step is one of the most important steps in the regulation of gene expression. Inducible (or repressible) genes, whose expression is varied in response to extra- or intracellular stimuli, often have complex promoters with upstream regulatory sites that help them respond to various stimuli (1). In contrast, the constitutively expressed genes have relatively simple promoters with invariant levels of gene expression.

It has been proposed relatively recently that the transcriptional activator responsive form of RNA polymerase II, the holoenzyme, includes the core polymerase II subunits, the general transcription factors, and a complex variously called the mediator, upstream stimulatory activity, or coactivators (1, 2). The mediator-coactivator complex is an important although not essential component of the holoenzyme (1, 3). In fact, a number of different holoenzymes have been proposed to coexist; each one of which is able to respond only to a subset of activators/regulators (4). It is increasingly becoming apparent that the subunits of the core polymerase may also play an active role in gene regulation by determining the interactions with either the mediators/coactivators or transcriptional activators (5-8).

The core RNA polymerase II of Saccharomyces cerevisiae is composed of twelve subunits, ten of which are essential for survival and five are shared between the three RNA polymerases (1). Rpb4, one of the non-essential subunits, is unique to eukaryotic RNA polymerase II in that it does not have a counterpart in the other two eukaryotic RNA polymerases (1, 9). It forms a subcomplex with one of the essential subunits, Rpb7, within the core RNA polymerase II. Biochemical analyses have revealed that the lack of the Rpb4 subunit also leads to the lack of the Rpb7 subunit in the immunoprecipitated polymerase (10, 11). The polymerase lacking Rpb4 and Rpb7 is inefficient in carrying out GAL1 promoter-directed transcription in vitro (10). Many groups including our own have shown recently that overexpression of RPB7 allows rescue of some of the phenotypes associated with the deletion of RPB4 (12-14). Rpb4 has been proposed to have a role in survival under stress conditions, in yeast (15).

We show here that RPB4 affects transcriptional activation of a subset of genes. This effect on activated transcription is much more pronounced than its effect on constitutive transcription. We also show here that this defect in activated transcription can be overcome by overexpression of transcriptional activators but not by increased recruitment of the TATA-binding protein (TBP)¹ to the promoter. Rpb7, the interacting partner of Rpb4, when overexpressed, partially rescues temperature sensitivity and some heat shock element-driven transcription but not all activated promoters.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Strains and Media-- The yeast strains used in this study were SY10-1: Mat a, his3200, ura3-52, leu2-3, 112, lys2, rpb4::HIS3/ pPS2; SY10-2: Mat a, his3-200, ura3-52, leu2-3, 112, lys2, rpb4::HIS3/pNS114; SY10-3: Mat a, his3200, ura3-52, leu2-3, 112, lys2, rpb4 :: HIS3/pPS4; SY10-4: Mat a, his3200, ura3-52, leu2-3, 112, lys2, rpb4::HIS3/pNS118; SY21-1: Mat a, ade2-1, his3200, ura3-52, leu2-3,112, trp1901, rpb4::HIS3/pPS2; SY21-2: Mat a, ade2-1, his3200, ura3-52, leu2-3,112, trp1901, rpb4::HIS3/pNS114; SY23-1: Mat , his3200, ura3-52, leu2-3,112, lys2, trp1901, cyh2^r, rpb4::HIS3/pPS2; SY23-2: Mat , his3200, ura3-52, leu2-3,112, lys2, trp1901, cyh2^r, rpb4::HIS3/pNS114. All the plasmids were transformed and amplified in Escherichia coli strain DH5 [supE44 lacU169, (80 lacZM15) hsdR17 recA1 gyrA96 thi-1 relA1]. The plasmids used in this study are described in Table I. Common media used for routine growth of yeast cultures and the manipulations of DNA were made as described (16). The yeast transformations were carried out by a modified lithium acetate method, which does not involve heat shock of the yeast cells (17).

Construction of Plasmids-- The construction of -galactosidase fusions to P_PHO5, heat shock element (HSE), P_INO1, and LexAop (LexA operator) have been previously described (18-21). The 3.9-kb EcoRI fragment from pPS76 containing the actin--galactosidase fusion was cloned downstream of the P_CUP1, P_GPD1, and P_TEF2 at the EcoRI site of pPS79, pPS190, and pPS189 to generate pNS98, pNS132, and pNS166 respectively (22, 23). To generate the P_ADH1--galactosidase fusion in pNS99, the 3.9-kb EcoRI fragment from pPS76 was blunt-ended with Klenow fragment of DNA Polymerase I (New England Biolabs) and cloned into the blunt-ended, HindIII-digested pPS31. pPS31 is identical to pGAD424 (CLONTECH). pPS121, bearing a P_GAL10--galactosidase fusion and pPS79 were gifts from Dr. U. Vijayaraghavan. pPS231 was a kind gift from P. J. Bhatt.

The 0.5-kb RPB7 open reading frame (ORF) was cloned under the P_TEF2 in pPS189 as a BamHI-XhoI fragment and further the P_TEF2-RPB7 fragment was subcloned into the SacI-KpnI sites of pPS5 (24) to generate pNS185. The HindIII site in the multiple cloning site of the plasmid pPS2 (24) was destroyed by end-filling it with the Klenow fragment of DNA polymerase I (New England Biolabs) and religation. Into this vector, pBP211, the RPB4 gene was cloned as a 1.2-kb BamHI-SalI fragment to generate pBP192. The rpb4-2 allele (pBP212) was generated by religating HindIII-digested pBP192 after blunt-ending with the Klenow fragment of DNA polymerase I. This generates an in-frame STOP codon leading to a deletion of the C-terminal 24 amino acids of Rpb4.

The 0.9-kb INO2 ORF was PCR-amplified from yeast genomic DNA using the following primers: Forward Primer (5'-CCCGGATCCTGCAACAAGCA-3'); Reverse primer (5'-CCCGTCGACTCAGGAATCATCC-3') and cloned as a fusion to the GAL4 activation domain (GAL4AD) under the P_ADH1 in pPS31. The 0.7-kb SPT15 ORF encoding TBP was PCR-amplified using the primers: 5'-CCGAATTCATGGCCGATGAGGAACG-3' and 5'-CGGGATCCCCTTCCCCATCACA-3' and cloned as an in-frame fusion to the LexA DNA binding domain (DBD) in pPS139 (25). The P_ADH1-LexA-DBD-TBP-T_ADH1 was transferred from this plasmid as a SphI fragment into the SphI-digested pPS30 (identical to pGBT9, CLONTECH) to obtain the LexA·TBP fusion in an appropriate vector (pBP215).

Temperature Sensitivity Test-- All strains analyzed for temperature sensitivity were first streaked onto plates containing synthetic complete (SC) medium + 2% glucose + 2% agar. Cells were restreaked at equal densities onto plates containing SC medium + 2% glucose + 2% agar. The plates were incubated at 25 °C, 34 °C, or 37 °C for 3 days before they were photographed.

-Galactosidase Assays-- The promoter activities of different constitutive and activated promoters in rpb4 and RPB4 strains were analyzed after growing the appropriate strains under the following growth conditions. GAL1 and GAL10: strains were grown in SC medium + 2% glucose until mid-log phase and then subcultured into SC medium + 2% galactose for 3-5 h (inducing condition). INO1: strains were grown in SC medium + 2% glucose until mid-log phase before they were assayed (derepressing condition). PHO5: strains were grown in SC medium + 2% glucose until mid-log phase and subcultured into SC medium without phosphate (derepressing condition) or with 7.5 mM ammonium phosphate. HSE: strains were grown in SC medium + 2% glucose until mid-log phase and one-half volume of culture was given a heat shock at 37 °C for 1 h (inducing condition). CUP1: strains were grown in SC medium +2% glucose until mid-log phase and subcultured to SC medium with (inducing condition) or without 100 µM CuSO₄.

The assay for the activity of constitutive promoters tested, P_ADH1, P_GPD1, and P_TEF2, was performed by growing the strains in SC medium +2% glucose until mid-log phase. Cells from both rpb4 and RPB4 strains were pelleted and -galactosidase assays were performed by using the glass bead method as described previously (26).

RNA Isolation and Northern Analysis-- For analysis of the CUP1 and GAL1 endogenous transcript levels, the strains were grown as described above. Total RNA was isolated as previously described (27). RNA samples were quantitated by measuring the absorbance at 260 nm, and equal amounts of RNA from rpb4 and RPB4 strains grown under non-inducing and inducing conditions were run on a 1% agarose-formaldehyde gel. RNAs were blotted on nitrocellulose membrane according to standard protocols (16). The 1.7-kb BamHI-SalI fragment from pPS232 (28), containing the entire GAL1 ORF and the 0.9-kb BamHI-SalI fragment from pPS80 (29), containing the entire CUP1 ORF were used for generating probes for analyzing endogenous levels of GAL1 and CUP1 genes, respectively. A 0.5-kb region of 18 S rRNA was PCR-amplified from the plasmid pNS245 and used for generating radiolabeled probes. [-³²P]dATP (PerkinElmer Life Science Products, BLU512H)-labeled DNA probes were generated using a random-primed DNA labeling kit (Amersham Pharmacia Biotech, RPN1604). The protocol suggested by the manufacturer was strictly followed. Routine hybridization and posthybridization procedures were followed. The blots were subjected to phosphor imaging analysis using a Fuji Phosphor Imager.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Activated Transcription Is Defective in a Strain Deleted for RPB4-- Activation of transcription from yeast promoters involves complex interactions between the upstream activating sequence (UAS)-bound transcriptional activator, the mediator/coactivator complexes, and the core RNA polymerase II, which leads to increased recruitment of the transcription-competent complex to the nearby promoter (1). Recent reports have suggested that the smaller subunits of core RNA polymerase II, Rpb3 and Rpb5, may act as contact points for transcriptional activators (5, 6). We have analyzed the role of another small subunit of RNA polymerase II, Rpb4, in activated transcription. Earlier reports suggest a role for Rpb4 in transcription initiation based on the inability of the RNA polymerase II purified from rpb4 cells to initiate transcription from a UAS GAL1-CYC1 promoter in vitro (10). We have used several well studied yeast promoters fused to the -galactosidase reporter gene and tested their activities in the absence and presence of RPB4. The -galactosidase activities were measured from cells grown in synthetic medium for the three constitutive promoters. For the six regulated promoters, the -galactosidase activities were measured from cells grown under inducing and non-inducing conditions. We observed that the activities of the promoters of the constitutive genes ADH1, TEF2, and GPD1 (from plasmids pNS99, pNS166, and pNS132 respectively) were reduced by 1.3-3-fold in rpb4 as compared with wild type (Fig. 1a). This suggests that the constitutive promoters tested are not significantly affected by the absence of RPB4.

View larger version (46K): [in this window] [in a new window]   Fig. 1.   The presence of RPB4 affects activated transcription from several activated promoters very severely but constitutive promoters are relatively unaffected. All the promoters except PADH1- and PCUP1--galactosidase fusions were tested in the strains SY10-1 (rpb4) and SY10-2 (RPB4) transformed with the appropriate plasmids. The PADH1 activity was tested in the strains SY10-3 (rpb4) and SY10-4 (RPB4) and PCUP1 activity was tested in the syngenic strain SY21-1 (rpb4) and SY21-2 (RPB4) because of lack of appropriate selection markers in the strain SY10. The rpb4 and RPB4 strains with the appropriate promoter-reporter fusions are represented as rpb4 and RPB4. The -galactosidase activities reflecting the promoter strengths are expressed as -gal units in bar graphs. The results represented are averages of three independent experiments performed with three transformants of each strain. Standard deviations are indicated by error bars. The inducing and non-inducing conditions and probes used are described under "Experimental Procedures." a, the constitutive promoters of ADH1, TEF2, and GPD1 genes were tested from the respective promoter--galactosidase reporter plasmids, pNS99, pNS166, and pNS132 in rpb4 and RPB4 strains. b, the five activated promoters from the genes GAL1, GAL10, INO1, PHO5, and the promoter-containing Heat shock element (HSE) were tested in rpb4 and RPB4 strains under non-inducing () and inducing (+) conditions. The ratio of the -galactosidase activity in RPB4 to that in rpb4 strains under non-inducing (U) and inducing conditions (I) is represented on top of each bar graph. For INO1 promoter fusions, no detectable level of -galactosidase activity was found in non-inducing conditions. Hence, only the inducing condition results are represented. c, the activated promoter of the CUP1 gene was tested in rpb4 and RPB4 strains under non-inducing () and inducing (+) conditions. d, the endogenous transcript level of the GAL1 and CUP1 genes were determined in rpb4 and RPB4 strains under non-inducing () and inducing (+) conditions using Northern analysis. The 18 S rRNA levels were used as loading controls.

Of the six regulated promoters tested, five promoters of the genes GAL1, GAL10, INO1, PHO5, and a promoter containing HSE (from plasmids pPS231, pPS121, pPS24, pPS122, and pPS111) showed highly inefficient regulated expression in the absence of RPB4 (Fig. 1b). The ratio of the -galactosidase activities in the wild-type strain to that in rpb4 has been calculated under inducing (I) and non-inducing (U) conditions and is represented on the top of each bar graph. It is obvious that the lack of RPB4 results in a drastic reduction in promoter activity under inducing conditions (from 14-320-fold depending on the promoter) but the activity under non-inducing conditions is affected to a much lesser extent in each case. This indicates that the reduced induction level is not just a reflection of reduced basal/uninduced levels of the promoter activity. On the other hand, the sixth inducible promoter tested, P_CUP1 (from the plasmid, pNS98) showed a reduction in activity of only about 3-fold in a rpb4 strain as compared with a wild-type strain even under inducing conditions (Fig. 1c).

Using another approach to study the transcriptional activity from the induced promoters, we tested the endogenous expression levels of some of the genes in rpb4 and RPB4 strains. The steady-state mRNA levels of CUP1 are similar in rpb4 and RPB4 strains even under inducing conditions (Fig. 1d). The RPB4 strain shows a drastic increase in the mRNA levels of the GAL1 transcript under inducing conditions as compared with the mRNA levels in non-inducing conditions. However, the GAL1 transcript is hardly visible in the rpb4 strain even under inducing conditions. This pattern of defective expression in rpb4 strain as compared with the RPB4 strain was also seen for the PHO5 and INO1-activated promoters (data not shown).

In summary, the results from the -galactosidase assays and the Northern analysis suggest that Rpb4 is involved in activated transcription. In the absence of Rpb4, transcription from constitutive promoters is less efficient, but the activated transcription from several promoters is severely compromised. These results also suggest that regulation of CUP1 transcription does not involve Rpb4 and is mechanistically different from other activated promoters (see "Discussion").

Overexpression of Rpb7 Specifically Rescues Defects in Heat Shock Promoter Activity in the Absence of Rpb4-- Previously we have shown that some of the stress phenotypes associated with the absence of RPB4 are partially rescued by overexpression of its interacting partner, RPB7 (12). This suggests that Rpb4 stabilizes the interaction of Rpb7 with the rest of the polymerase. The results presented above suggest a role for Rpb4 in activated transcription. We analyzed the effect of high levels of RPB7 on the induced promoter activities of GAL10 and INO1 and the HSE-containing promoter (derived from the SSA1 gene), in the absence of RPB4. Because the TEF2 promoter is constitutively expressed at high levels and we have seen that it is not significantly affected by the absence of RPB4 (Fig. 1a), we overexpressed RPB7 from the TEF2 promoter on a multicopy plasmid (pNS185) in the rpb4 strain SY10-1. We have confirmed that the transcript levels of RPB7 expressed from this construct are several-fold higher as compared with the rpb4 parent strain and that this overexpressed level of RPB7 could rescue the temperature-sensitive phenotype of the parent strain at 37 °C (data not shown). At 37 °C, the activity of the HSE containing promoter was approximately 8-fold higher than the activity in rpb4 (Fig. 2a). On the other hand, as shown in Fig. 2b, -galactosidase activities from the P_GAL10 and P_INO1 promoter-reporter fusions in rpb4+P_TEF2-RPB7 strain are very similar to their activities in the rpb4 strain. This suggests that overexpression of RPB7 in rpb4 strain can rescue the defect in activated transcription from the HSE-containing promoter but not from the GAL10 and INO1 promoters.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Overexpression of RPB7 partially rescues the activation defect of rpb4 at some promoters. The effect of overexpression of RPB7 on the activation defect of rpb4 strains was assayed using heat shock element-containing promoter (a) and GAL10, INO1 (b). The results presented are average values obtained with three transformants of each strain. Standard deviations are indicated by error bars. rpb4 strain with the appropriate promoter-reporter plasmid carrying either vector, PTEF2-RPB7 or RPB4 (represented as rpb4, RPB7, and RPB4, respectively) were assayed for the HSE, GAL10, and INO1 promoter activities. The inducing and non-inducing conditions used are described under "Experimental Procedures."

Defects in Activated Transcription Are Partially Compensated by Overexpression of the Cognate Transcriptional Activators-- It was earlier observed that the purified polymerase lacking both subunits, Rpb4 and Rpb7, was defective in promoter-specific transcription initiation in vitro, which could be rescued partially by the addition of a potent chimeric transcriptional activator (10). Because we found that in vivo- (unlike the in vitro result) activated transcription is significantly affected in the absence of RPB4, we decided to test if overexpressed cognate activators will have a rescuing effect on the specific-activated promoters. We overexpressed two specific transcriptional activators, GAL4 and INO2, from the ADH1 promoter (30) and tested the effect on the activity of the GAL10 and INO1 promoters, respectively, in the absence of RPB4. Both the activators when expressed at higher than normal levels partially rescued the activation defect of rpb4 (Fig. 3). In the case of GAL4 overexpression, the uninduced levels in rpb4 are also significantly higher (see "Discussion," Ref. 31). Under inducing conditions, the GAL10 promoter activity is even higher than under non-inducing conditions, indicating that the rescue of activated transcription in rpb4 is specific to overexpression of the activator. To test whether the effect of overexpression of transcriptional activator is specific to the cognate regulated promoter and not a nonspecific general effect on transcription, we also tested the promoter activities in the presence of an excess of the non-cognate transcriptional activators. As shown in Fig. 3, the activated promoters are affected by overexpression of only the cognate activator. This confirmed that the effect of the activators is specific and not an effect on transcription in general.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Overexpression of activators rescues the activation defect of rpb4 specifically but partially. The activators of GAL10 and INO1 genes, GAL4 and INO2, respectively, were overexpressed from PADH1 in a multicopy plasmid. Results presented are averages of values obtained with three transformants each and the standard deviations are represented by error bars. The rpb4 strain with the appropriate promoter-reporter plasmid carrying either vector or activators (GAL4/INO2) or RPB4 are represented as rpb4, GAL4, INO2, and RPB4, respectively. The -galactosidase assays were performed under inducing conditions (+) and non-inducing conditions () as described under "Experimental Procedures."

The observation that activated transcription is inefficient in the absence of Rpb4, and overexpression of activators partially rescues the activation defect of rpb4 strains could be explained by direct or indirect contact between activators and Rpb4. Previously, the mammalian transcriptional activator, EWS-Fli, has been shown to interact directly with the human homolog of Rpb7 (8). We used the directed two-hybrid assay to test whether there is a physical interaction between either Rpb4 or Rpb7 and the GAL4 and INO2 activators. Neither of the activators interacted with either of the subunits (data not shown).

Removal of a C-terminal Region of RPB4 Leads to Defective Activated Transcription-- The comparison of the Rpb4 protein sequence (using the PRODOM data base) across species shows that the protein has a conserved C-terminal region whereas the N-terminal sequence appears unique to the S. cerevisiae protein (32). The 150 amino acid long domain, 16118 (as numbered in the PRODOM data base) is shared by all the homologs in this alignment and has several invariant residues (Fig. 4a) (32). Using a convenient HindIII restriction site we decided to alter the invariant sequence PSL to PS (STOP). This manipulation deletes the remaining 24 amino acids from the C terminus of Rpb4, eight of which are very highly conserved (identical in four or more of the six homologs compared). We tested if this change in the sequence affects the role of RPB4. We observed that the mutant rpb4-2 is defective in rescue of temperature sensitivity of the rpb4 strain even at 34 °C (Fig. 4b). The deletion mutant was as defective as the rpb4 strain in activation from the promoter of the GAL10 gene (Fig. 4c). We have tested transcript levels corresponding to the RPB4 gene both in RPB4 and the rpb4-2 carrying strains and found them comparable (data not shown). We could not formally rule out the possibility that the loss of the C-terminal part of the protein affects protein stability because even wild-type expression levels of Rpb4 were below the limits of detection.

View larger version (84K): [in this window] [in a new window]   Fig. 4.   The C-terminal conserved region of Rpb4 is essential for its function. a, the alignment of all known homologs of Rpb4 using the program PRODOM Version 2000.1 identified a conserved C-terminal domain, 16118. The invariant residues in the 150 amino acid long domain are indicated in white with black background and those identical in at least four sequences are in black with gray background. The residues deleted from the S. cerevisiae RPB4 allele, rpb4-2, are underlined. b, the rpb4-2 allele does not rescue the temperature-sensitive phenotype of rpb4. rpb4 strain (SY10) transformed with either Vector, RPB4, or rpb4-2 were assayed for temperature sensitivity. rpb4 strain with vector, or rpb4-2 allele, or RPB4 are represented as rpb4, rpb4-2, and RPB4, respectively. c, the C-terminal 24 amino acids are essential for the activation function of Rpb4. The strains as described in b were transformed with the PGAL10-LacZ plasmid and were assayed for the activity of the PGAL10 under non-inducing () and inducing (+) conditions. The results presented are averages of three independent experiments with three transformants, and the standard deviations are represented by error bars.

LexA·TBP-mediated Recruitment of the Holoenzyme Is Not Sufficient for Activated Levels of Transcription in the Absence of RPB4-- Because the absence of RPB4 causes a severe defect in the activation of several activated genes, which can be partially rescued by the overexpression of activators, we wanted to further analyze the nature of the defect. It has been shown earlier that several components of the holoenzyme when tethered to a strong DNA binding domain can circumvent the requirement for activators (33, 34). Many transcriptional activators act by stabilizing the interaction of TBP with the promoter (35, 36). Other workers have shown that the requirement for transcriptional activators can be circumvented by artificially tethering TBP using a LexA·TBP fusion protein (33). The LexA protein binds strongly to the LexAop element positioned upstream of the TATA box, thus stabilizing TBP-promoter interaction. If Rpb4 plays a role in the interaction of transcriptional activators with the transcription machinery, we proposed that artificial recruitment by tethering of TBP might circumvent the need for Rpb4. We used a reporter plasmid carrying eight copies of the LexAop upstream of a TATA box, which drives expression of -galactosidase gene downstream. We introduced the LexA·TBP fusion and the -galactosidase reporter under the LexAop control into rpb4 and RPB4 strains. The LexA·TBP fusion failed to activate the expression of the reporter plasmid in a rpb4 strain. However, the presence of the RPB4 gene allowed activation through the LexA·TBP fusion (Fig. 5). We also tested the activation potential of the LexA·TBP fusion in the presence of the C-terminal-deleted RPB4, the rpb4-2 mutant and found the mutant to be defective similar to the deletion strain (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Synthetic activation by promoter-tethered TBP requires the presence of RPB4. The strains SY23-1 (rpb4) and SY23-2 (RPB4) transformed with the LexAop-LacZ plasmid (pPS144) and the LexA DBD-TBP fusion plasmid, pBP215, were assayed for -galactosidase activity. The results presented are averages of three independent experiments done with three transformants each and their standard deviations are indicated by error bars. rpb4 strain with RPB4 and rpb4 strain with vector are represented as RPB4 and rpb4.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Over the last decade, there has been a tremendous increase in our understanding about mechanisms of activation of transcription in eukaryotes, with major contributions coming from the yeast system. It has been reported that the transcription machinery exists in the form of a holoenzyme comprising RNA polymerase II, the general transcription factors, and the mediator/coactivator complex. An important concept that has emerged is that the holoenzyme is the form of polymerase competent to respond to activators (1, 2). The composition of the holoenzyme differs substantially depending on the purification method employed and today it is accepted that these differences may actually reflect in vivo differences (4). In most cases, the differences have been found to be in the ancillary factors and components of the mediator/coactivator complexes. Until recently, the core RNA polymerase II was considered an integral part of all holoenzymes. Since then it has been reported that subunits Rpb3 and Rpb5 of the yeast core polymerase actually affect the activation of subsets of genes showing that core subunits of the RNA polymerase II can have regulatory roles. Core RNA polymerase II subunits may determine the composition of the holoenzyme, probably through the interactions with other components, in turn affecting the activation of a subset of genes.

As reported here, Rpb4 falls into the category of those core subunits, which affect activation of a subset of genes. Choder et al. (15) have shown that rpb4 is defective in stress response based on its inability to survive under extreme temperatures and in stationary phase. Brendel and Karlin (37) have proposed that Rpb4 may have activator-like features based on sequence analysis. Edwards et al. (10) employing P_GAL1 as a test promoter had reported almost a decade ago that in vitro, the Rpb4-Rpb7 subcomplex affects promoter-dependent initiation whereas Rosenheck and Choder (38) suggested that Rpb4 somehow allows RNA polymerase II to transcribe at extreme temperatures in vitro. While this manuscript was in preparation, Tan et al. (39) reported that Rpb4 is required for basal transcription in in vitro assays whereas in vivo studies show that basal and activated transcription are both defective. Our in vivo results show that the constitutive promoters are not significantly affected in the absence of RPB4 (Fig. 1a). We have clearly shown using both promoter -galactosidase fusions and Northern analyses of steady-state levels of endogenous transcripts that the absence of RPB4 affects many if not all of the activated transcription units tested (Fig. 1b). The only activated promoter tested that is not defective in the absence of RPB4 is P_CUP1 (Fig. 1, c and d). This promoter is also known to be one of the few yeast promoters that does not require the holoenzyme for activation suggesting that the CUP1 gene is activated by alternative mechanisms (40, 41). Therefore, it is not entirely surprising that Rpb4 is dispensable for activation of CUP1. The activation defect is also shown by a nonsense mutant lacking a small, highly conserved 24-amino acid stretch at the C terminus of the protein.

Several investigators have reported that Rpb4 and Rpb7 subunits form a subcomplex. The close proximity of these two proteins within the core polymerase has been demonstrated using electron microscopic analysis of crystals of RNA polymerase II (42). Our own studies earlier showed that overexpression of its interacting partner, RPB7, compensates for the lack of RPB4 in the temperature sensitivity assay suggesting that one of the functions of Rpb4 is to stabilize the interaction of Rpb7 with the rest of the polymerase (12). Rpb7 has also been identified by Tan et al. (39) as a multicopy suppressor of temperature sensitivity of rpb4. It also partially restored the steady-state levels of transcripts from various non-heat shock genes whose expression was affected in rpb4. However, in our studies using promoter-reporter fusions, we do not find a similar rescue. This difference implies that Rpb7 overexpression increases the steady-state levels of transcripts, probably through a post-transcriptional effect. Sro9p another suppressor reported by Tan et al. (39) has also been shown to rescue rpb4 by increasing mRNA stability. That the overexpression of RPB7 rescues the HSE activity is in accordance with the above result, and similar observations reported by others (Fig. 2) (12-14). This observation suggests that the two subunits may have different roles in expression of stress and non-stress-related genes.

To investigate further the role of RPB4 in activation of transcription, we decided to test if the defect in activation is because of defective interaction, either direct or indirect, with some of the activators. In either case, then excess of transcriptional activator may be able to rescue the defect. It was indeed seen that overexpression of the cognate activators partially rescued activation from the corresponding promoters. Overexpression of Gal4 results in relatively high levels of transcription under non-induced conditions. This is most likely because of the fact that the constitutively expressed Gal4 is present in excess over the negative regulator Gal80 thereby releasing active Gal4 even under non-inducing conditions (31). The fact that under inducing conditions the level of transcription is significantly higher than under non-inducing conditions suggests that the overexpression is indeed rescuing activated transcription even in the absence of Rpb4. The human homolog of Rpb7 has been shown to interact directly with transcriptional activators (8). We have seen that activators do not interact directly with Rpb4 even though their overexpression rescues the transcriptional activation defect of rpb4. The effect of Rpb4 on activation is probably mediated through other proteins.

Previous reports show that transcriptional activation in the absence of true activators is possible if one tethers TBP to a strong DNA binding domain and recruits it to the promoter (33, 34). In vivo, TBP occupancy at promoters increases on activation of the gene (35, 36). These reports have suggested that at least one of the ways in which activators function is by increasing the recruitment of the holoenzyme to the promoter. Because we find that the tethered TBP was ineffective in activation in the absence of RPB4, we conclude that the holoenzyme recruitment through tethering of TBP is not sufficient for activation in the absence of this subunit. In summary, our results show that RPB4 plays an important role in activation of a subset of genes in the model eukaryote, S. cerevisiae. This function is not shared directly by its interacting partner, Rpb7.

	ACKNOWLEDGEMENTS

We thank Drs. E. Golemis, U. Vijayraghavan, L. Nover, J. Bhat, S. Chavez, and T. Platt for gifts of various plasmids used in this study. We thank Drs. V. Sarangdhar, C-M. Chiang, and the members of our laboratory for encouragement and useful discussions.

	FOOTNOTES

^* This work was supported by grants from the Dept. of Science and Technology and the Council for Scientific and Industrial Research, India (to P. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 91-80-309-2292; Fax: 91-80-360-2697; E-mail: pps@mcbl.iisc.ernet.in.

Published, JBC Papers in Press, May 29, 2001, DOI 10.1074/jbc.M010952200

	ABBREVIATIONS

The abbreviations used are: TBP, TATA-binding protein; HSE, heat shock element; kb, kilobase; ORF, open-reading frame.

	REFERENCES

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