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Molecular and Cellular Biology, September 1998, p. 5364-5370, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Requirement for a Functional Interaction between Mediator Components Med6 and Srb4 in RNA Polymerase II Transcription

Young Chul Lee and Young-Joon Kim*

Laboratory of Molecular Genetics, Center for Molecular Medicine, Samsung Biomedical Research Institute, Kangnam-ku, Seoul 135-230, Korea

Received 3 April 1998/Returned for modification 25 May 1998/Accepted 9 June 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Regulated transcription of class II genes of the yeast Saccharomyces cerevisiae requires the diverse functions of mediator complex. In particular, MED6 is essential for activated transcription from many class II promoters, suggesting that it functions as a key player in the relay of activator signals to the basal transcription machinery. To identify the functional relationship between MED6 and other transcriptional regulators, we conducted a genetic screen to isolate a suppressor of a temperature-sensitive (ts) med6 mutation. We identified an SRB4 allele as a dominant and allele-specific suppressor of med6-ts. A single missense mutation in SRB4 can specifically suppress transcriptional defects caused by the med6 ts mutation, indicating a functional interaction between these two mediator subunits in the activation of transcription. Biochemical analysis of mediator subassembly revealed that mediator can be dissociated into two tightly associated subcomplexes. The Med6 and Srb4 proteins are contained in the same subcomplex together with other dominant Srb proteins, consistent with their functional relationship revealed by the genetic study. Our results suggest not only the existence of a specific interaction between Med6 and Srb4 but also the requirement of this interaction in transcriptional regulation of RNA polymerase II holoenzyme.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The mediator of RNA polymerase II (Pol II) is required for diverse aspects of the transcription process, such as activation, repression, basal transcription, and phosphorylation of the C-terminal repeat domain (CTD) of the largest Pol II subunit (1, 9, 12). Genetic and biochemical studies identified more than 20 polypeptides as the mediator components, including Ssn-Srb family proteins (5, 13, 19, 28), Gal11, Rgr1, Sin4, and Rox3 (4, 7, 17, 25-27), and Med1 to Med8 (16, 18, 21). Studies of these mediator subunits revealed that some mediator genes are genetically required only in the regulation of specific genes, whereas others are necessary for general transcription by Pol II in vivo. Although these results suggest that several groups of mediator subunits and their interactions with Pol II are essential for regulated transcription of target genes, experimental evidence illustrating functional interactions among these groups in the mediation of transcriptional regulation is lacking.

Our previous study of MED6 revealed that it is required for transcriptional activation of many but not all genes (16). These findings suggest that Med6 is a key player in signal relay from activators to the basal transcription machinery. On the other hand, SRB genes were identified as suppressors of the CTD truncation mutation, and these proteins are thus regarded as mediator components that are situated near Pol II (5, 19, 23, 28). The global effect of the srb4 temperature-sensitive (ts) mutation on Pol II transcription (29) suggests that Srb4 is required for general, rather than regulated, transcription through its interaction with the CTD of Pol II. Although the SRB2 and SRB5 genes are dispensable for cell viability, in vitro transcription assays using nuclear extracts from deletion mutant strains reveals that Srb2 and Srb5 have important roles in basal transcription (11, 29).

To delineate the functional relations among the mediator subunits, especially between the mediator subgroups involved in either general or regulated transcription, we examined the genetic and biochemical interactions among the various mediator components. Here we report the identification of SRB4 as a dominant suppressor of the med6 ts mutation, as well as a biochemical analysis of mediator assembly that reveals a tight association among mediator components with similar functions.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation of a dominant extragenic suppressor of the med6-ts2 mutation. Saccharomyces cerevisiae yeast strains and plasmids used in this study are listed in Table 1 and 2, respectively. Yeast strain YCL44, in which the MED6 gene was replaced by the med6-ts2 gene (designated med6 ts in reference 16) on plasmid pRS316, was mutagenized by treatment with 1% ethyl methanesulfonate as described elsewhere (10). Mutagenized cells were incubated on yeast extract-peptone-dextrose (YPD) plates at 37°C for 4 days, and colonies capable of growth at 37°C were isolated. Among these isolates, intragenic suppressors were removed by replacing pRS316-med6ts2 in each strain with pRS313-med6ts2 via the plasmid shuffle method (24). To isolate dominant suppressors, each putative extragenic suppressor strain was crossed with the opposite mating-type med6 mutant strain YCL51 [Mata (pRS316-med6-ts2)], and the resulting diploid strain was tested for temperature sensitivity at 37°C. Diploids that grew at 37°C were sporulated, and the growth of each spore at 37°C was examined. The diploids that gave two viable spores at 37°C were isolated as dominant extragenic suppressors by a single-gene mutation.

                              
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  		TABLE 1.   Yeast strains used in this study

                              
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  		TABLE 2.   Plasmids used in this study

Construction of a suppressor genomic library and suppressor gene cloning. To clone a dominant suppressor gene, genomic DNA from the mixed culture of dominant suppressor strains was prepared and digested partially with Sau3AI, and DNA fragments larger than 4 kb were cloned into the BamHI site of the pRS316 vector. From a total of 3 × 105 Escherichia coli transformants, library plasmids were prepared and transformed into the med6-ts2 strain YCL8. One hundred thousand transformants were incubated at 30°C for a day, moved to 37°C, and allowed an additional 3-day incubation to isolate colonies that grew at the restrictive temperature. To confirm that suppression of the ts phenotype was dependent on the transformed genomic DNA, the library plasmid from each putative suppressor clone was recovered and retransformed into YCL8 to test its ability to suppress the ts phenotype. The genomic inserts of the suppressor plasmids were sequenced, and an open reading frame that overlapped in the inserts was regarded as a putative suppressor gene. Its genuineness was confirmed with the gene fragment obtained by in vivo gap repair (20) of the putative suppressor gene. The suppressor mutation was determined by sequencing both strands of the suppressor gene obtained from the library and from in vivo gap repair with the use of synthetic primers.

RNA preparation and analysis. Exponentially growing yeast cells were divided into two aliquots and allowed to grow for another 2.5 h at either 30 or 37°C. Total RNA and poly(A)+ RNA were prepared from each population of frozen cells and used for S1 nuclease protection analysis and Northern blot analysis, respectively (16). Oligonucleotide probes used in S1 analyses (for DED1 and GAL1) and antisense RNA probes used in Northern analyses (for MFalpha 1, MATalpha 1, and PYK1) were identical to those used in a previous study (16).

Immunoblot analysis with whole-cell extracts. Yeast cells were grown at 30°C in YPD media (15 ml) to late exponential phase. The cell pellets were washed with water, resuspended in 0.5 ml of 3× lysis buffer (30% glycerol [9]), and disrupted in a Bead-Beater (Biospec) with the same volume of glass beads (0.5-mm diameter; Sigma). This and all subsequent steps were performed at 4°C. Cell lysates were filtered to remove glass beads and cleared by centrifugation at 15,000 × g for 15 min. To the supernatant, 1/10 volume of 4 M potassium acetate (pH 7.6) and 1/100 volume of 10% polyethyleneimine (pH 8.0) were added. The mixture was shaken gently for 30 min and then centrifuged at 15,000 × g for 15 min to obtain a whole-cell extract. When necessary, the whole-cell extract was concentrated by the addition of saturating amounts of ammonium sulfate. Immunoblot analysis was performed with 30 µg of whole-cell extract and polyclonal antisera against mediator components.

Immunoprecipitation. Anti-Rgr1 antiserum was generated in a rat, using as the antigen a recombinant six-histidine-tagged Rgr1 protein fragment (the N-terminal 460 amino acids) purified from E. coli. Affinity-purified anti-Med6 antibody (150 µg) (16) and crude anti-Rgr1 antiserum (200 µl) were conjugated with protein A-agarose (200 µl) and protein G-agarose (200 µl) beads (GIBCO/BRL), respectively, as described elsewhere (17). Each aliquot of antibody beads (20 µl) was incubated for 6 to 12 h at 4°C with 10 µg of RNA Pol II holoenzyme (holo-Pol II) (MonoQ fraction), washed three times with 400 µl of IP (immunoprecipitation) buffer-150 (16) containing various concentrations of urea, and finally washed with IP buffer-150. The bound proteins were eluted with 100 mM glycine (pH 3.5), precipitated with 10% trichloroacetate, and analyzed by silver staining of sodium dodecyl sulfate (SDS)-polyacrylamide gels or immunoblotting. Holo-Pol II was purified as described previously (9) from S. cerevisiae YSJ1, in which MED6 was replaced by a six-histidine-tagged MED6 gene on pRS313.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation of a suppressor for the med6-ts2 mutation. To understand the mechanism by which mediator components, particularly Med6, relay a signal from activators to the basal transcription machinery, it is necessary to establish the functional relationship of Med6 with other transcriptional regulators. As a first step toward this goal, we screened suppressors of the med6-ts2 mutation on the basis of its ts phenotype as described in Materials and Methods. Out of 100 million mutagenized med6-ts2 cells, we isolated 24 extragenic suppressors together with 100 intragenic suppressors. The ts viability of diploids, obtained by mating individual extragenic suppressors with the opposite mating type of the med6-ts2 mutant, revealed 9 recessive and 15 dominant suppressors. Among them, only six recessive and five dominant suppressors produced two viable spores at 37°C, suggesting that a single mutation was involved in the suppression activity of each strain.

A positive screen at 37°C with a mixed genomic library prepared from the five dominant suppressor strains allowed isolation of two extragenic suppressor clones (YCL69 and YCL70) from 100,000 transformants screened. DNA sequencing of the inserts of the two clones and database searches with these sequences revealed that the two independent suppressor clones contained in common the SRB4 gene. Sequence analyses of these suppressor SRB4 alleles identified a single missense mutation that changed Glu286 to Lys in both clones. We named this dominant suppressor allele SRB4-101. To identify the suppressor strain from which SRB4-101 originated, we examined the SRB4 suppressor mutation in each of the suppressor strains. Because the mutation in SRB4-101 changed the gene sequence GAATC to AAATC, wild-type and suppressor SRB4 alleles were distinguishable by the presence of a HinfI restriction site only in the wild-type allele. The HinfI restriction analysis of the SRB4 fragments amplified from the med6-ts2 and suppressor strains showed that the HinfI site was lost in all of the dominant suppressor strains that we isolated (Fig. 1A).


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  		FIG. 1.   Suppression phenotypes of the SRB4-101 mutation. (A) All five dominant suppressors contained the SRB4-101 mutation. A 473-bp fragment of the SRB4 gene containing an SRB4-101 mutational site was amplified by PCR from the med6-ts2 strain (YCL8; lane 1) and five dominant suppressor strains (SME27, -120, -122, -123 [identical to YCL50], and -124; lanes 2 to 6, respectively). The amplified DNA was digested with HinfI and resolved on a 2% agarose gel. Two DNA fragments of 171 and 156 bp were produced from wild-type SRB4 (lane 1) by the presence of an internal HinfI site (GAATC). The SRB4-101 mutation caused the generation of a 327-bp DNA fragment (lanes 2 to 6), as a result of the absence of the HinfI site (AAATC). (B) Dominant phenotype of SRB4-101 suppression. Yeast strains were spotted in duplicate on YPD agar plates, and each plate was incubated for 3 days at either the permissive (30°C) or nonpermissive (37°C) temperature. The temperature sensitivities of the wild type (YCL10), med6-ts2 mutant (YCL8), med6-ts2 mutants containing the SRB4-101 allele (YCL50, -81, -70, and -77), and SRB4-101 suppressor mutants in a MED6 wild-type background (YCL71 and -78) were compared. The chromosomal copy of the SRB4 gene was deleted in strains YCL77 and -78. The YCL strains used in the spot assay are represented by the YCL numbers at the corresponding positions in the box, and their genotypes are described in Table 1.

To confirm whether the G-to-A mutation described above is truly responsible for suppression of med6-ts2, we retrieved the SRB4 allele from the suppressor strain YCL50 by in vivo gap repair and tested its suppression activity. As shown in Fig. 1B, the YCL81 strain containing the retrieved SRB4 allele as an extra copy grew as well as did YCL50 and YCL70 at the restrictive temperature. On the other hand, the introduction of a wild-type SRB4 gene into YCL8 as an extragenic copy had no effect on its ts phenotype (data not shown). These results suggest that the suppression of med6-ts2 was conferred by the G-to-A mutation in the SRB4-101 allele. Although the SRB4-101 mutation rescues the ts phenotype of the med6-ts2 strain, it had no detectable phenotype by itself under various growth conditions (Fig. 1B [YCL71 and -78] and data not shown). In addition, the suppression of the med6-ts2 mutation by SRB4-101 was not enhanced by the removal of the wild-type SRB4 gene from the YCL70 strain (YCL77 [Fig. 1B]), implying that this suppression is achieved by the selective and specific interactions between the two mutations.

Restoration of the transcriptional defects in the med6-ts2 mutant by SRB4-101 mutation. To test whether the suppression of the med6 ts phenotype resulted from the suppression of transcriptional defects, we examined the effect of SRB4-101 on the crippled transcriptional activation in the med6-ts2 mutant in vivo. As shown in Fig. 2A, the inability of the med6-ts2 mutant (YCL8) to display GAL1 transcriptional activation (2.5-fold induction) was rescued by the suppressor mutation (up to 40% of the wild-type level; 14-fold). However, SRB4-101 alone in a wild-type MED6 genetic background (YCL78) had no effect on GAL1 transcriptional regulation. Restoration of transcriptional defects was observed consistently, as in the transcription of MFalpha 1 and PYK1 (Fig. 2B); SRB4-101 caused 3.5- and 2-fold increases in the transcription of MFalpha 1 and PYK1, respectively. The observed up-regulation of transcription in vivo by SRB4-101 was specific only to those genes affected by the med6-ts2 mutation. SRB4-101 had no effect on the transcription of genes that do not require MED6 activity (for example, MATalpha 1 and DED1) or on GAL1, PYK1, and MFalpha 1 transcription when wild-type MED6 was present (Fig. 2, YCL78). In addition, the SRB4-101 mutant (YCL78) showed no difference from the wild type (YCL10) in many of the aspects of transcription that we tested (for example, transcription of MED6 and HIS3). These results indicate that the observed suppression of transcriptional defects did not result from a nonspecific enhancement of total cellular activity caused by SRB4-101. Rather, it is a suppressor mutation in SRB4 that specifically rescued the med6 mutational defects.


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  		FIG. 2.   Restoration of the transcriptional defects in the med6-ts2 mutant by SRB4-101. (A) Suppression of GAL1 activation defect. Total RNA was prepared from wild-type (W; YCL10), med6-ts2 (T; YCL8), med6-ts2 SRB4-101 (S; YCL77), and SRB4-101 (O; YCL78) strains grown under the indicated conditions and used for S1 protection assays to measure the amounts of repressed (YPGlc [YP plus glucose]) and activated (YPGal [YP plus galactose]) GAL1 transcripts. The DED1 gene, whose transcription is not affected by med6-ts2 mutation, was used as an RNA loading control. (B) Effects of SRB4-101 mutation on MFalpha 1 and PYK1 transcriptional defects. Poly(A)+ RNA was prepared from the indicated yeast strains cultured at either the permissive (30°C) or nonpermissive (37°C) temperature and hybridized with the probes shown at the left to analyze their transcript levels. The abbreviations are as in panel A. The levels of DED1 transcripts are also shown as an internal control.

Allele specificity of SRB4-101 suppression. Mutation site analysis of med6-ts2 revealed that functional defects as well as a reduced amount of the mutant Med6 protein (med6p) (16) were required to cause the growth defect at 37°C (15). Therefore, dominant suppression of the med6-ts2 defect could be accomplished by increasing the binding affinity of holo-Pol II to med6p or by rescuing the functional defects of med6p. Suppression by SRB4-101 appears to occur by the later mechanism, because no difference was detectable between med6-ts2 (YCL8) and suppressor (YCL77) strains in the amount of med6p present in the whole-cell extract as well as in the immunoaffinity-purified holo-Pol II fraction (Fig. 3A).


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  		FIG. 3.   Allele specificity of SRB4-101 suppression. (A) The reduced amount of med6p was not recovered by SRB4-101 mutation. (Left) Immunoblot analysis was performed with the antisera against the indicated proteins to reveal their contents in whole-cell extracts (30 µg of protein) of wild-type (YCL10), med6-ts2 (YCL8), med6-ts2 SRB4-101 (YCL77), SRB4-101 (YCL78), med6-ts1 (YCL90), and med6-ts6 (YCL91) cells. (Right) The holo-Pol II fraction was immunopurified from whole-cell extracts (1 mg of protein) of the indicated strains (with the use of anti-Rgr1 antibody beads) and subjected to immunoblot analysis. (B) Suppression of the ts phenotype by SRB4-101 is specific to med6-ts2 allele. Temperature sensitivities of med6 ts strains transformed with SRB4-101 gene (YCL93 and -95) were compared with those of their parental strains (YCL90 and -91). Wild-type (YCL10), med6-ts1 (YCL90 and -93), and med6-ts6 (YCL91 and -95) were spotted with 10-fold serial dilutions onto duplicate YPD plates, which were incubated for 3 days at either 30 or 37°C. (C) SRB4-101 rescues the transcriptional defect of med6-ts2 allele specifically. Total RNA was prepared from the indicated YCL strains grown under the indicated conditions (YPD [YP plus glucose] or YPGal [YP plus galactose]) and used for S1 protection assays to measure the amounts of activated GAL1 transcripts. The DED1 transcripts were shown as an RNA loading control. The number above each lane in panels A and C represents the YCL strain of the corresponding number.

SRB4-101 is not a bypass suppressor because Med6 was required for the viability of the suppressor strain (data not shown). To examine the allele specificity of this requirement, we tested the suppression activity of SRB4-101 in two additional med6 ts mutants, one (med6-ts1) with a change of Phe31 to Ser (F31S) and the other (med6-ts6) with seven amino acid changes (L28P, K47T, T134A, Q171R, T177M, M273L, and I275V). Although these med6 alleles showed different mutational profiles, they all caused a similar transcriptional defect and a reduced amount of med6p in med6-ts1 (YCL90) and med6-ts6 (YCL91) cells (Fig. 3A and C and data not shown). However, unlike med6-ts2, SRB4-101 did not rescue the ts phenotypes (Fig. 3B, YCL93 and -95) or the transcriptional defects (Fig. 3C) of med6-ts1 and -ts6 cells. This allele specificity of SRB4-101 suppression indicates a specific functional interaction between MED6 and SRB4.

Separation of mediator into two stable subcomplexes. The specific genetic interaction between SRB4-101 and med6-ts2 strongly suggested a direct physical interaction between the two corresponding proteins in the mediator complex. We examined this possibility with a number of approaches both in vitro (column binding assay, far-Western analysis, and UV cross-linking analysis) and in vivo (yeast two-hybrid assay) but failed to detect any significant interaction between these proteins (data not shown). These negative results suggest that the interaction between Med6 and Srb4 proteins may require additional mediator components. To test this idea, we investigated subcomplex assembly of mediator by high-salt treatment of immunoprecipitated holo-Pol II. To identify the mediator subunits associated with Med6, holo-Pol II was immobilized on the anti-Med6 antibody agarose beads, and the polypeptides retained after urea wash were analyzed. After 1 M urea wash, most of the Med3, -4, -7, -8, and -9, and Srb7 proteins were removed from the Med6-anchored beads, whereas Rox3 and Srb2, -4, -5, and -6 proteins remained tightly associated with the beads (Fig. 4A, lanes 6 and 7). The large mediator proteins (Rgr1, Gal11, Sin4, and Med1) were found in the Med6-anchored beads after the wash, but the amounts detected were significantly less than those of the other Med6-associated proteins.


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  		FIG. 4.   Dissociation of mediator complex into two stable subcomplexes. (A) Differential dissociation of mediator components by urea treatment. Holo-Pol II (MonoQ fraction) was immunoprecipitated with anti-Rgr1 antibody (lanes 2 to 5) or anti-Med6 antibody (lanes 6 and 7) beads, washed with buffers containing no urea (lanes 2 and 6) or 1 M (lanes 3 and 7), 2 M (lane 4), or 3 M (lane 5) urea, and visualized by silver staining. The mediator components that remained on the antibody beads after 1 M urea treatment are indicated at the right of each panel; the positions of core-Pol II (c-polII) subunits (lane 1) are indicated at the left. Since the holo-Pol II was prepared from a strain in which wild-type MED6 was replaced with six-histidine-tagged MED6 (Med6*), the Med6* comigrates with Med4 in an SDS-gel. Therefore, the amounts of Med6* and Med4 in each subcomplex lane are half of the amount of proteins marked with Med6* and Med4 in the total lane. (B) Silver-stained SDS-polyacrylamide gel of intact mediator complex (Total; immunopurified with anti-Rgr1 antibody beads), Rgr1-containing subcomplex (Rgr1-sub; proteins retained by holo-Pol II immobilized on anti-Rgr1 antibody beads that were washed with 2 M urea), and Med6-containing subcomplex (Med6-sub; 2 M urea eluate from Rgr1-immobilized holo-Pol II immunoprecipitated with anti-Med6 antibody beads in the presence of 1 M urea). The components of Rgr1- and Med6-containing subcomplexes are indicated on the left and right, respectively. The low abundance of Med6 in the Med6 immunoprecipitate is due to an incomplete elution of Med6 from the antibody beads under the elution conditions used in this experiment. A stoichiometric amount of Med6 in the immunoprecipitate was detected when the bead-bound proteins were directly loaded on a protein gel without elution. (C) Immunoblot analysis of the intact mediator (Total) and Rgr1-containing (Rgr1-sub), and Med6-containing (Med6-sub) subcomplexes with antisera specific to mediator components indicated at the right.

We examined these physical interactions among the mediator subunits in the opposite direction with the use of more stringent conditions. Because Rgr1, Gal11, Sin4, and Med3 (p50) proteins have been shown to interact with each other (6, 17), holo-Pol II was immobilized on the anti-Rgr1 antibody agarose, and the polypeptides retained after an extensive urea wash were analyzed (Fig. 4A, lanes 2 to 5). A 2 M urea wash removed Med6, Rox3, and Srb2, -4, -5, and -6 proteins along with core-Pol II, and a 3 M urea wash caused a further loss of Sin4 and Med3, -4, -7, -8, and -9 proteins from the Rgr1-anchored holo-Pol II. As a result, an Rgr1 subcomplex containing Gal11, Med1, and Srb7 proteins was finally retained on the antibody beads.

Therefore, the results from the dissociation experiments suggest that there are two mediator subcomplexes, each consisting of specific, tightly associated components. To test directly whether the mediator components that were dissociated from holo-Pol II existed as individual proteins or as subcomplexes, a 2 M urea eluate from Rgr1-anchored holo-Pol II was immunoprecipitated with anti-Med6 antibody beads. As shown in Fig. 4B, all of the dissociated proteins (Srb2, -4, -5, and -6, Med6, and Rox3) were coimmunoprecipitated as a complex in the presence of high salt, indicating that mediator can be separated into two stable subcomplexes: (i) an Rgr1-containing subcomplex (Rgr1 plus Gal11, Sin4, Srb7, and Med1, -3, -4, -7, -8, and -9) and (ii) a Med6-containing subcomplex (Med6 plus Rox3 and Srb2, -4, -5, and -6). These results were confirmed by immunoblot analysis with the use of antisera directed against mediator subunits (Fig. 4C). The coexistence of Med6 and Srb4 in the same mediator subcomplex and the genetic interaction identified from our suppressor study suggest a physical and functional interaction between the two proteins.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Although studies conducted previously on the functions of MED6 and SRB4 were based on their mutant phenotypes (3, 16, 29), no direct functional relationship between them was noted. In contrast, our identification of SRB4 as an allele-specific dominant suppressor of med6-ts2 suggests a specific functional interaction between the Med6 and Srb4 proteins. This result also indicates that interacting mediator subunits must act in concert to carry out the various transcriptional processes and that their functional cooperation is important for appropriate transcriptional regulation. Srb4 appears to be a good candidate for a central regulatory component at the core of this functional integration. Because the defects in both general transcription machinery (CTD) and activation-specific machinery (MED6) were corrected by distinct SRB4 alleles (data not shown), the connection of functionally different mechanisms via Srb4 seems to be important in transcriptional activation by holo-Pol II.

The genetic interaction between MED6 and SRB4 was supported further by the biochemical analysis of mediator subassembly. Even though we subjected the mediator to conditions harsh enough to disrupt most known protein-protein interactions, two multicomponent subcomplexes of the mediator remained tightly associated. The composition of the Med6-associated subcomplex (Srb2, -4, -5, and -6 and Rox3) indicated a putative interaction between Med6 and Srb4. Because the lack of Srb2 and -5 proteins did not cause a dissociation of Med6 from the mutant holo-Pol II and vice versa (14, 16), the interaction between these proteins appears not to be essential for the formation of a Med6-associated subcomplex. Therefore, one of the remaining polypeptides, Srb4, Srb6, and Rox3, may provide the primary affinity to hold the Med6 protein in this subcomplex. Considering the genetic results described previously, not to mention of the size of the polypeptide, Srb4 seems to be the best candidate for such a role.

The cofractionation of all Srb proteins, which behave as dominant suppressors of CTD truncation mutation, suggests that the primary function of this subcomplex lies in the modulation of Pol II activity via CTD interaction. The finding of Med6 and Rox3 along with other Srb proteins is surprising, because their mutant phenotypes with respect to transcriptional regulation are similar to those of mediator subunits in the Rgr1-associated subcomplex (2, 4, 6-8, 16, 22, 25, 27). These proteins, along with Srb7, which is the only recessive CTD suppressor (6) belonging to the Rgr1-associated subcomplex, may behave as bridges of regulation signal transfer between the two subcomplexes.

The significance of these interactions within the mediator complex and the putative structure of mediator assembly were demonstrated further by the analysis of the Rgr1-associated subcomplex. Li et al. (17) observed that holo-Pol II from the Rgr1 C-terminal truncation mutant was devoid of Gal11, Sin4, and Med3 (p50), suggesting that these polypeptides associated with holo-Pol II through the C-terminal region of Rgr1. In addition to these mediator polypeptides, our experiments revealed that Rgr1 is also tightly associated with Med1, -4, -7, -8, and -9 and Srb7 proteins. This result indicates that Med1, -4, -7, -8, and -9 and Srb7 must interact with Rgr1 through regions other than the C-terminal domain. The binding strength of each polypeptide exhibited here indicates that the interaction of Med1 and Srb7 with Rgr1 is more primary than that of other proteins (Fig. 4A, lane 5). However, whether these polypeptides form a module with a specific regulatory function, as does Gal11, Sin4, and Med3 subassembly, must be examined by genetic and biochemical analyses of their mutants. The direct interaction between Gal11 and Rgr1 that we propose here differs from the model by Li et al. (17), which suggested a chain of contacts from Rgr1 via Sin4 to Gal11. This discrepancy may be a result of destabilization of the interaction between Gal11 and Rgr1 in the absence of Sin4 in vivo. The significance of this interaction has been well documented by the similar genetic phenotypes observed in gal11, sin4, and rgr1 deletion mutants (2, 6, 8, 17, 22). Therefore, the identification of another group of mediator subunits (Med1, -4, -7, -8, and -9 and Srb7) that associate with Rgr1 suggests that these polypeptides may have a similar but distinct function. We proposed a model for putative structure on mediator subassembly in Fig. 5 on the basis of genetic and biochemical evidence presented here and by others.


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  		FIG. 5.   Model for the global structure of mediator assembly. Mediator can be dissociated into two stable subcomplexes (one Rgr1 associated and one Med6 containing) by urea treatment. The Rgr1-associated subcomplex is composed of Rgr1 plus Gal11 module subunits (Gal11, Sin4, and Med3), Srb7, Med1, and other Med subunits (Med4, -7, -8, and -9). The Med6-containing subcomplex is composed of dominant Srb subunits (Srb4, -2, -5, and -6) plus Med6 and Rox3 proteins. Involvement of Gal11 module subunits in positive and negative regulations of transcription suggests their role as transducers in the relay of signals from activators, repressors, or other unknown transcriptional regulators. Association of Srb7 and other Med subunits (Med1, -4, -5, -7, and -9) with Rgr1 was not affected by the truncation of C-terminal region of Rgr1 (14, 17), indicating that these subunits may form a distinct module interacting with other regulatory factors. On the other hand, the association of all of the dominant Srb proteins in a subcomplex suggests that the Med6-containing subcomplex is situated near Pol II, thus modulating Pol II activity via CTD interaction. No information regarding the interacting surface between the two subcomplexes is available at present, but Med6, Rox3, and Srb7 proteins are thought to behave as bridges of signal transfer between the two subcomplexes, considering their distinct mutant phenotypes (e.g., all of these subunits are essential for cell viability [see Discussion]).

The functional differences of mediator components revealed by genetic and biochemical analyses suggest that a subset of mediator proteins may be responsible for each of the mediator activities. We have tried without success to identify the function of each subcomplex in a defined in vitro system. The lack of mediator subcomplex activity may be a result of severe inhibitory effects of the harsh conditions applied during the dissociation procedure. Therefore, reconstitution with recombinant forms of mediator components may be required to reveal the functional roles of the subcomplexes. This approach should provide a detailed description of subunit assembly and functional specificity, which will aid in the delineation of mechanisms of transcriptional regulation by a mediator.

    ACKNOWLEDGMENTS

We thank Soyoung Min and Sang Jun Han for helpful discussions and Kelly LaMarco for careful reading of the manuscript. We also thank Juri Kim for the help with suppressor isolation and Richard Young, Andres Aguilera, and Toshio Fukasawa for providing antibodies.

This work was supported by grants from SBRI (B-96-004) and Republic of Korea Ministry of Health and Welfare (HMP-97-B-3-0030 of the 1997 Good Health R&D project) to Y.-J.K.

    FOOTNOTES

* Corresponding author. Mailing address: Laboratory of Molecular Genetics, Center for Molecular Medicine, Samsung Biomedical Research Institute, 50 Ilwon-dong, Kangnam-ku, Seoul 135-230, Korea. Phone: 82-2-3410-3638. Fax: 82-2-3410-3649. E-mail: yjkim{at}smc.samsung.co.kr.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Molecular and Cellular Biology, September 1998, p. 5364-5370, Vol. 18, No. 9
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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