COMMUNICATION:
Identification of Rox3 as a Component of Mediator and RNA Polymerase II Holoenzyme*

(Received for publication, August 15, 1996, and in revised form, October 22, 1996)

Claes M. Gustafsson Dagger §, Lawrence C. Myers Dagger §par , Yang Li Dagger **, Michael J. Redd Dagger Dagger , Mary Lui §§, Hediye Erdjument-Bromage §§, Paul Tempst §§ and Roger D. Kornberg Dagger

From the Dagger  Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305-5400, the Dagger Dagger  Department of Biochemistry and Biophysics, University of California San Francisco, California 94143-0502, and the §§ Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Yeast Rox3 protein, implicated by genetic evidence in both negative and positive transcriptional regulation, is identified as a mediator subunit by peptide sequence determination and is shown to copurify and co-immunoprecipitate with RNA polymerase II holoenzyme.


INTRODUCTION

Transcription reconstituted with a set of pure general transcription factors and RNA polymerase II from the yeast Saccharomyces cerevisiae is unresponsive to activator proteins (1). Addition of a multiprotein complex termed mediator enables a response to activators and also stimulates both basal transcription and phosphorylation of the C-terminal domain of the polymerase (1). Mediator interacts with the C-terminal domain, forming a polymerase holoenzyme, whose existence first came to light from genetic studies of the Srb family of proteins and their isolation in a complex with polymerase II (2). Srbs were subsequently identified as subunits of mediator, along with about a dozen additional polypeptides (1). Current work is directed toward the nature of these polypeptides and their roles in transcription in vitro and in vivo.

Three mediator polypeptides have been identified as products of the GAL11, SIN4, and RGR1 genes, previously recovered from disparate genetic screens for mutations affecting transcription (3, 4, 5). The SIN4 and RGR1 screens were for loss of repression, pointing to a role of mediator in negative regulation of transcription. It has since emerged that Gal11, Sin4, and Rgr1 all participate in control of the same large family of genes and that they are involved in both activation and repression (6, 7, 8, 9). Consistent with the genetic findings, Gal11, Sin4, and Rgr1 appear to interact in a subassembly of the mediator complex (10). These studies, together with effects of srb mutations on INO1 gene expression (11), provide evidence for a role of mediator in transcriptional regulation in vivo.

Mediator polypeptides in addition to Srbs and the Gal11 subassembly have been designated Med1-Med8 (12). Elsewhere we and others describe the isolation and characterization of MED6, a novel gene important for inducible transcription and required for yeast cell growth.12 Here we report on Med8 which, like the members of the Gal11 subassembly, proves to be the product of a gene previously characterized as important for transcriptional regulation in vivo.


MATERIALS AND METHODS

Protein Purification

Purification of RNA polymerase II holoenzyme from the hemagglutinin antigen-tagged Rox3 strain was as described (12).

Protein Sequencing

Peptides were generated from the PVDF3-bound, 31-kDa protein by tryptic digestion in situ (13, 14) and were fractionated by reversed phase HPLC (15) with the use of a 1-mm Reliasil C18 column. Selected peak fractions were analyzed by a combination of automated Edman chemical degradation (16) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (13, 17). Peptide sequences were compared with entries in the Saccharomyces Genome Data base (SGD, Stanford Genomic Resources, Stanford University) with the use of the National Center for Biotechnology Information (NCBI) BLAST program (18). Experimental masses of these and additional peptides were compared with the theoretical average isotopic masses of fragments expected to result from tryptic digestion of the identified proteins (with the use of PeptideSearch software; Dr. Matthias Mann, EMBL, Heidelberg, Germany).

Construction of Hemagglutinin Antigen-tagged Rox3 Strain

The 3'-end (240 base pairs) of the ROX3 gene was amplified by polymerase chain reaction and ligated in frame with the hemagglutinin tags of pMR46, a derivative of Yplac111 CEN ARS LEU2 in which CEN and ARS have been deleted and three copies of the hemagglutinin tag (HA-tag) have been inserted in the polylinker (19). The resulting plasmid was digested with XbaI and transformed into Saccharomyces cerevisiae BJ5459 (MATa ura3-52 trp1 lys2-801 leu2-D1 his3 D200 pep4::HIS3 prb1 D1.6R can1) (20). Integration at the ROX3 locus resulted in a hemagglutinin-tagged full-length copy of the gene and another partial copy consisting of the 3'-most 240 base pairs of the gene with no promoter.

Immunoprecipitation

Anti-Med6 antibodies were coupled to protein A-Sepharose beads and used to immunoprecipitate purified Rox3HA holopolymerase as described (10) with the following modifications. Subsequent to coupling and prior to incubation with Rox3HA holopolymerase fraction, 50 µl of anti-Med6 beads were washed with 100 µl of a buffer containing 50 mM glycine, pH 2.3, 150 mM NaCl, followed by several washes with 100 µl of IP-200 buffer (20 mM HEPES, pH 7.6, 10% glycerol, 12.5 mM MgCl2, 0.1 mM EDTA, 0.2% Nonidet P-40, 0.1 mM dithiothreitol, 200 mM potassium acetate). Rox3HA holopolymerase Mono Q fraction 50 (10 µl) was diluted with 40 µl of buffer Q, 0.5 (25 mM Tris acetate, pH 7.8, 10% glycerol, 1 mM EDTA, 0.5 M potassium acetate) and sedimented for 5 min at 13,000 rpm prior to incubation with the beads. Following incubation with the diluted fraction for 4 h at 4 °C, the beads were washed three times with 200 µl of IP-200 buffer and eluted twice for 10 min at room temperature with 17.5 µl of 5 M urea. To the combined eluates was added 20 µl of 2 × SDS loading buffer (20% glycerol, 10% 2-mercaptoethanol, 4.6% SDS, 125 mM Tris-Cl, pH 6.8, 0.1% (w/v) bromphenol blue) for SDS-PAGE and immunoblotting.


RESULTS

The occurrence of Rox3 in mediator preparations was revealed by peptide sequencing. Mediator polypeptides were resolved by SDS-PAGE and transferred to a PVDF membrane. A band just below the 31-kDa marker, designated Med8 (12), was excised and subjected to tryptic digestion. The partial sequence of one fragment, SYPSEFANQNQGGAQAPFDIDDLAF, corresponded identically with residues 148-172 of the amino acid sequence expected from the ROX3 gene. The experimental mass of this peptide was in good agreement (within 0.01% of) the theoretical average isotopic mass of the expected tryptic peptide containing the partial sequence. Mass analyses allowed accurate positioning of several more tryptic peptides in the Rox3 protein sequence.

The occurrence of Rox3 in RNA polymerase II holoenzyme was confirmed by copurification and co-immunoprecipitation. A derivative of the ROX3 gene, denoted ROX3HA, was constructed for expression of the protein with three copies of the influenza virus hemagglutinin antigen appended to the C terminus. ROX3HA was able to replace ROX3, so it evidently supplies all the essential functions of the wild type gene.4 RNA polymerase II holoenzyme was purified from a ROX3HA strain by chromatography on Bio-Rex 70, DEAE-Sephacel, hydroxylapatite, and Mono Q, followed by gel filtration through Bio-Sil SEC 400, as described (12). The gel filtration step was performed at high ionic strength to minimize nonspecific protein-protein interactions. Immunoblots revealed comigration of epitope-tagged Rox3 with holoenzyme components Rpb1, Gal11, and Srb4 on both Mono Q and Bio-Sil SEC 400 (Fig. 1, A and B).


Fig. 1. Copurification of hemagglutinin antigen-tagged Rox3 with RNA polymerase II holoenzyme. A, comigration of Rpb1, Srb4, and Rox3 on Mono Q. Fractions around the peak of holopolymerase (750 mM potassium acetate) were analyzed by immunoblotting with antisera against Rpb1, Srb4, and the hemagglutinin antigen. B, comigration of Srb4, Gal11, and Rox3 on Bio-Sil SEC 400. Fractions were analyzed by immunoblotting with antisera against Srb4, Gal11, and the hemagglutinin antigen.
[View Larger Version of this Image (35K GIF file)]


Immunoprecipition was performed with antibodies against the Med6 component of mediator/holoenzyme coupled to protein A-Sepharose. Incubation of holopolymerase with the antibody-Sepharose and subsequent washing steps were performed under stringent conditions. Immunoblot analyses demonstrated that Rox3HA and holoenzyme components Srb4, Rpb1, and Rpb3 were almost entirely bound by the antibody beads (Fig. 2). We conclude that all Rox3 in the holopolymerase preparation was associated with holoenzyme.


Fig. 2. Co-immunoprecipitation of Gal11, Srb4, Rpb3, and Rox3HA from Mono Q fraction 50 by anti-Med6 antibody coupled to protein A-Sepharose. Bands from the immunoprecipitate (pellet) are weaker than from the starting fraction (load) due to loss of holopolymerase under the stringent washing conditions.
[View Larger Version of this Image (24K GIF file)]


The reciprocal co-immunoprecipitation experiment was performed with antibodies against the influenza hemagglutinin antigen of Rox3HA. SDS-PAGE and silver staining showed that RNA polymerase II holoenzyme was bound by the antibody beads (data not shown), though the extent of binding was not determined.

The relative amount of Rox3 in RNA polymerase II holoenzyme was estimated from the A214 of tryptic peptides resolved by HPLC and by SDS-PAGE, staining with Coomassie Blue, and densitometry. The intensity of staining of the Med8/Rox3 band, relative to those of the other mediator/holoenzyme components Gal11, Rgr1, Med1, Med7, and Srb7, was consistent with expectation. We conclude that Rox3 is stoichiometric with other components of mediator/holoenzyme.


DISCUSSION

The results presented here demonstrate the occurrence of Rox3 in mediator and RNA polymerase II holoenzyme. Immunoprecipitation experiments and quantitation establish that Rox3 is an integral rather than a trace or substoichiometric component of holoenzyme preparations. Genetic studies indicate that the association of Rox3 with mediator/holoenzyme is physiologically relevant and not an artifact of biochemical isolation. The ROX3 gene was originally found in a search for mutants leading to overexpression of the heme-regulated CYC7 gene (21). ROX3 proved to be essential for cell growth, and Rox3 protein was shown to be nuclear localized. ROX3 was later identified as SSN7 (22), derived from a screen for mutants bypassing the requirement for Snf1 protein kinase for expression of glucose-repressed genes (23). ROX3 is also synonymous with RMR1, whose mutation can relieve glucose repression of the CYB2 gene (24). Finally, ROX3 is needed for full induction of the GAL1 gene in the presence of galactose (24). And Rox3 fused to the DNA-binding domain of lexA stimulates transcription from a promoter with a lexA-binding site (22). ROX3 therefore plays a role in both repression and activation of transcription.

In its dual regulatory capacity, Rox3 resembles the previously described mediator proteins Gal11, Sin4, and Rgr1 (see Introduction). Like Rox3, these proteins first came to light from genetic screens unconnected with RNA polymerase II. Their subsequent identification as components of mediator/holoenzyme, together with that of Rox3, provides the strongest evidence to date for a role of mediator in transcriptional regulation in vivo.

Through its involvement in transcription of the CYC7 gene, induced under all forms of cellular stress (25, 26, 27), Rox3 contributes to the global stress response. Truncation of Rox3 diminishes the CYC7 response to heat shock and osmotic stress, and deletion of RTS1, a multicopy supressor of mutations in ROX3, causes a similar phenotype (25). RTS1 encodes a cytoplasmic protein highly homologous to the B'-subunit of human protein phosphatase 2A, which is involved in several intracellular signaling pathways (28). It has been suggested that Rts1 is part of such a pathway, affecting gene expression in response to stress (25). The genetic relationship between RTS1 and ROX3 raises the possibility of mediator/holoenzyme as an end point of this pathway.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grant GM36659 (to R. D. K), by National Science Foundation Grant BIR-940123 (to P. T.), by National Cancer Institute Core Grant 5 P30 CA08748 to the Sloan-Kettering Sequencing Laboratory, and by National Institutes of Health Grant GM37049-10 (to A. D. Johnson). 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.
§   These authors contributed equally to this work.
   Recipient of a Swedish Cancer Society postdoctoral fellowship.
par    Supported by Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship DRG-1361.
**   Supported by a Bank of America-Giannini Foundation Fellowship for medical research in California.
1    Y. Li, S. Bjorklund, Y.-J. Kim, W. Lane, and R. D. Kornberg, submitted for publication.
2    Y.-C. Lee, S. Min, B. S. Gim, and Y.-J. Kim, submitted for publication.
3    The abbreviations used are: PVDF, polyvinylidene difluoride; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.
4    M. J. Redd and A. D. Johnson, unpublished observations.

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