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Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans, the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1. Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent transcriptional and morphological changes, suggesting that Ssn3 kinase activity negatively regulates Flo8.Under embedded conditions, when ssn3Δ/Δ and efg1Δ/Δ mutants were hyperfilamentous, FLO8 was essential for hypha formation. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that the ssn3Δ/Δ medium alkalinization phenotype, which is dependent on STP2, a transcription factor involved in amino acid utilization, also requires FLO8 and EFG1. Together, these data show that Ssn3 activity can modulate Flo8 and its direct and indirect interactions in different ways, and underscores the potential importance of considering Ssn3 function in the control of transcription factor activities., Author summary In Candida albicans, Ssn3 kinase activity co-regulates the transcription of numerous genes involved in hyphal growth, metabolism and nutrient acquisition, immune cell interactions, and drug resistance. Using a strain in which Ssn3 could be rapidly and selectively inhibited, we identified both known and novel Ssn3 targets. We identified two phosphosites in Flo8, a regulator of morphology and virulence, that were shown to negatively regulate Flo8 levels and activity. The data and tools presented here will enable a better understanding of how Ssn3 impacts transcriptional and post-transcriptional regulation in order to coordinate processes during physiological and morphological transitions as well as during steady state growth.

Mediator complex has recently emerged as an important regulator of gene expression in pathogenic fungi. Mediator is a multi-subunit complex of polypeptides involved in transcriptional activation in eukaryotes, with roles including preinitiation complex (PIC) assembly and chromatin remodeling. Within the last decade, Mediator has been shown to play an integral role in regulating virulence gene expression and drug resistance in human fungal pathogens. In some fungi, specific Mediator subunits have been shown to be required for virulence. In Candida species, duplication and expansion of Mediator subunit encoding genes has occurred on at least three occasions (CgMED15 in C. glabrata and MED2/TLO in C. albicans and C. dubliniensis) suggesting important roles for Mediator in the evolution of these pathogens. This review summarises recent developments in our understanding of Mediator in fungal pathogens and the potential for the development of therapeutic drugs to target Mediator functions.

Farnesol, a quorum-sensing molecule, inhibits Candida albicans hyphal formation, affects its biofilm formation and dispersal, and impacts its stress response. Several aspects of farnesol's mechanism of action remain incompletely uncharacterized. Among these are a thorough accounting of the cellular receptors and transporters for farnesol. This work suggests these processes are linked through the Zn cluster transcription factors Tac1 and Znc1 and their induction of the multidrug efflux pump Cdr1. Specifically, we have demonstrated that Tac1 and Znc1 are functionally activated by farnesol through a mechanism that mimics other means of hyperactivation of Zn cluster transcription factors. This is consistent with our observation that many genes acutely induced by farnesol are dependent on TAC1, ZNC1, or both. A related molecule, 1-dodecanol, invokes a similar TAC1-ZNC1 response, while several other proposed C. albicans quorum-sensing molecules do not. Tac1 and Znc1 both bind to and upregulate the CDR1 promoter in response to farnesol. Differences in inducer and DNA binding specificity lead to Tac1 and Znc1 having overlapping, but nonidentical, regulons. Induction of genes by farnesol via Tac1 and Znc1 was inversely related to the level of CDR1 present in the cell, suggesting a model in which induction of CDR1 by Tac1 and Znc1 leads to an increase in farnesol efflux. Consistent with this premise, our results show that CDR1 expression, and its regulation by TAC1 and ZNC1, facilitates growth in the presence of high farnesol concentrations in C. albicans and in certain strains of its close relative, C. dubliniensis.

Farnesol, a quorum-sensing molecule, inhibits C. albicans hyphal formation, affects its biofilm formation and dispersal, and impacts its stress response. Several aspects of farnesol’s mechanism of action remain incompletely uncharacterized. Among these are a thorough accounting of the cellular receptors and transporters for farnesol. This work suggests these themes are linked through the Zn cluster transcription factors Tac1 and Znc1, and their induction of the multi-drug efflux pump Cdr1. Specifically, we have demonstrated that Tac1 and Znc1 are functionally activated by farnesol through a mechanism that mimics other means of hyperactivation of Zn cluster transcription factors. This is consistent with our observation that many genes acutely induced by farnesol are dependent on TAC1, ZNC1, or both. A related molecule, 1-dodecanol, invokes a similar TAC1/ZNC1 response, while several other proposed C. albicans quorum sensing molecules do not. TAC1 and ZNC1 both bind to and up-regulate the CDR1 promoter in response to farnesol. Differences in inducer and DNA binding specificity lead to Tac1 and Znc1 having overlapping, but non-identical, regulons. TAC1 and ZNC1 dependent farnesol induction of their target genes was inversely related to the level of CDR1 present in the cell, suggesting a model in which induction of CDR1 by Tac1 and Znc1 leads to an increase in farnesol efflux. Consistent with this premise, our results show that CDR1 expression, and its regulation by TAC1 and ZNC1, facilitates growth in the presence of high farnesol concentrations in C. albicans, and certain strains of its close relative C. dubliniensis.

Long-term azole treatment of patients with chronic Candida albicans infections can lead to drug resistance. Gain-of-function (GOF) mutations in the transcription factor Mrr1 and the consequent transcriptional activation of MDR1 , a drug efflux coding gene, is a common pathway by which this human fungal pathogen acquires fluconazole resistance. This work elucidates the previously unknown downstream transcription mechanisms utilized by hyperactive Mrr1. We identified the Swi/Snf chromatin remodeling complex as a key coactivator for Mrr1, which is required to maintain basal and induced open chromatin, and Mrr1 occupancy, at the MDR1 promoter. Deletion of snf2 , the catalytic subunit of Swi/Snf, largely abrogates the increases in MDR1 expression and fluconazole MIC observed in MRR1 GOF mutant strains. Mediator positively and negatively regulates key Mrr1 target promoters. Deletion of the Mediator tail module med3 subunit reduces, but does not eliminate, the increased MDR1 expression and fluconazole MIC conferred by MRR1 GOF mutations. Eliminating the kinase activity of the Mediator Ssn3 subunit suppresses the decreased MDR1 expression and fluconazole MIC of the snf2 null mutation in MRR1 GOF strains. Ssn3 deletion also suppresses MDR1 promoter histone displacement defects in snf2 null mutants. The combination of this work with studies on other hyperactive zinc cluster transcription factors that confer azole resistance in fungal pathogens reveals a complex picture where the induction of drug efflux pump expression requires the coordination of multiple coactivators. The observed variations in transcription factor and target promoter dependence of this process may make the search for azole sensitivity-restoring small molecules more complicated.

The multisubunit eukaryotic Mediator complex integrates diverse positive and negative gene regulatory signals and transmits them to the core transcription machinery. Mutations in individual subunits within the complex can lead to decreased or increased transcription of certain subsets of genes, which are highly specific to the mutated subunit. Recent studies suggest a role for Mediator in epigenetic silencing. Using white-opaque morphological switching in Candida albicans as a model, we have shown that Mediator is required for the stability of both the epigenetic silenced (white) and active (opaque) states of the bistable transcription circuit driven by the master regulator Wor1. Individual deletions of eight C. albicans Mediator subunits have shown that different Mediator subunits have dramatically diverse effects on the directionality, frequency, and environmental induction of epigenetic switching. Among the Mediator deletion mutants analyzed, only Med12 has a steady-state transcriptional effect on the components of the Wor1 circuit that clearly corresponds to its effect on switching. The MED16 and MED9 genes have been found to be among a small subset of genes that are required for the stability of both the white and opaque states. Deletion of the Med3 subunit completely destabilizes the opaque state, even though the Wor1 transcription circuit is intact and can be driven by ectopic expression of Wor1. The highly impaired ability of the med3 deletion mutant to mate, even when Wor1 expression is ectopically induced, reveals that the activation of the Wor1 circuit can be decoupled from the opaque state and one of its primary biological consequences.

The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. Genome wide localization studies have demonstrated that Mediator occupancy not only correlates with high levels of transcription, but that the complex also is present at transcriptionally silenced locations. We provide evidence that Mediator localization is guided by an interaction with histone tails, and that this interaction is regulated by their post-translational modifications. A quantitative, high-density genetic interaction map revealed links between Mediator components and factors affecting chromatin structure, especially histone deacetylases. Peptide binding assays demonstrated that pure wild-type Mediator forms stable complexes with the tails of Histone H3 and H4. These binding assays also showed Mediator-histone H4 peptide interactions are specifically inhibited by acetylation of the histone H4 lysine 16, a residue critical in transcriptional silencing. Finally, these findings were validated by tiling array analysis that revealed a broad correlation between Mediator and nucleosome occupancy in vivo, but a negative correlation between Mediator and nucleosomes acetylated at histone H4 lysine 16. Our studies show that chromatin structure and the acetylation state of histones are intimately connected to Mediator localization.

In Saccharomyces cerevisiae, multiple approaches have arrived at a consensus TATA box sequence of TATA(T/A)A(A/T)(A/G). TATA-binding protein (TBP) affinity alone does not determine TATA box function. To discover how a minimal set of factors required for basal and activated transcription contributed to the sequence requirements for a functional TATA box, we performed transcription reactions using highly purified proteins and CYC1 promoter TATA box mutants. The TATA box consensus sequence is a good predictor of promoter activity. However, several nonconsensus sequences are almost fully functional, indicating that mechanistic requirements are not the only selective pressure on the TATA box. We also found that the effect of a mutation at a certain position is often dependent on other bases within a particular TATA box. Although activators and coactivators strongly influence TBP recruitment and stability at promoters, neither Mediator, the activator Gal4-V16, nor TFIID specifically compensate for the low transcription levels of the weak TATA boxes. The addition of Mediator to purified transcription reactions did, however, increase the functional selectivity for certain consensus TATA sequences. Transcription in whole-cell extracts or in vivo with these TATA box mutants indicated that factors, other than those in our purified system, may help initiate transcription from weak TATA boxes.

Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen.

Recent studies of fungal genomes have shown that subtelomeric regions of chromosomes are areas of rapid evolution that facilitate adaptation to novel niches [1]. Several years ago, analysis of the genome of the human pathogenic yeast Candida albicans revealed the presence of a large family of telomeric orfs (TLO genes) [2]. The function of this gene family remained an enigma in C. albicans genetics for many years; however, recent studies have revealed that the TLO genes encode a subunit of the Mediator complex with roles in transcriptional regulation [3,4]. This gene family expansion is unique to C. albicans, the species responsible for the majority of human yeast infections and the species that is most commonly recovered as a human commensal. If selective pressures in the host have driven this expansion, it is likely that this gene family somehow contributes to the success of C. albicans as a commensal and opportunistic pathogen. To support this hypothesis, it was first necessary to determine the exact function of Tlo proteins in C. albicans. Armed with this knowledge, investigators are now beginning to understand how possession of multiple copies of TLO could contribute to the virulence properties of C. albicans.

Candida albicans is an important human fungal pathogen in both immunocompetent and immunocompromised individuals. C. albicans regulation has been studied in many contexts, including morphological transitions, mating competence, biofilm formation, stress resistance, and cell wall synthesis. Analysis of kinase- and phosphatase-deficient mutants has made it clear that protein phosphorylation plays an important role in the regulation of these pathways. In this study, to further our understanding of phosphorylation in C. albicans regulation, we performed a deep analysis of the phosphoproteome in C. albicans . We identified 19,590 unique peptides that corresponded to 15,906 unique phosphosites on 2,896 proteins. The ratios of serine, threonine, and tyrosine phosphosites were 80.01%, 18.11%, and 1.81%, respectively. The majority of proteins (2,111) contained at least two detected phosphorylation sites. Consistent with findings in other fungi, cytoskeletal proteins were among the most highly phosphorylated proteins, and there were differences in Gene Ontology (GO) terms for proteins with serine and threonine versus tyrosine phosphorylation sites. This large-scale analysis identified phosphosites in protein components of Mediator, an important transcriptional coregulatory protein complex. A targeted analysis of the phosphosites in Mediator complex proteins confirmed the large-scale studies, and further in vitro assays identified a subset of these phosphorylations that were catalyzed by Cdk8 (Ssn3), a kinase within the Mediator complex. These data represent the deepest single analysis of a fungal phosphoproteome and lay the groundwork for future analyses of the C. albicans phosphoproteome and specific phosphoproteins.

In Saccharomyces cerevisiae, the RNA polymerase II (RNA Pol II) carboxyl-terminal domain (CTD) is required for viability, and truncation of the CTD results in promoter dependent transcriptional defects. A CTD-less RNA Pol II is unable to support transcription in yeast extracts, but basal transcription reactions reconstituted from highly purified general transcription factors are CTD-independent. To reconcile these two findings, we have taken a biochemical approach using yeast extracts and asked whether there is a factor in the cell that confers CTD-dependence upon transcription. By placing a cleavage site for the tobacco etch virus protease prior to the CTD, we have created a highly specific method for removing the CTD from RNA Pol II in yeast whole cell extracts. Using derivatives of this strain, we have analyzed the role of the Srb8-11 complex, Mediator, and TFIIH, in CTD-dependent basal transcription by either mutation or immunodepletion of their function. We have found that Mediator is a direct intermediary of CTD-dependent basal transcription in extracts and that the requirement for Mediator and the CTD in basal transcription originates from their ability to compensate for a limiting amount of TFIIH activity in extracts.

The basal transcription factor TFIID is composed of the TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). Although TBP alone binds to the TATA box of DNA and supports basal transcription, the TAFs have essential functions that remain poorly defined. In order to study its properties, TFIID was purified from Saccharomyces cerevisiae using a newly developed affinity tag. Analysis of the final elution by mass spectrometry confirms the presence of all the known TAFs and TBP, as well as Rsp5, Bul1, Ubp3, Bre5, Cka1, and Cka2. Both Taf1 and Taf5 are ubiquitinated, and the ubiquitination pattern of TFIID changes when BUL1 or BRE5 is deleted. Purified TFIID binds specifically to promoter DNA in a manner stabilized by TFIIA, and these complexes can be analyzed by native gel electrophoresis. Phenanthroline-copper footprinting and photoaffinity cross-linking indicate that TFIID makes extensive contacts upstream and downstream of the TATA box. TFIID supports basal transcription and activated transcription, both of which are enhanced by TFIIA.

The TLO genes are a family of telomere-associated ORFs in the fungal pathogens Candida albicans and C. dubliniensis that encode a subunit of the Mediator complex with homology to Med2. The more virulent pathogen C. albicans has 15 copies of the gene whereas the less pathogenic species C. dubliniensis has only two (CdTLO1 and CdTLO2). In this study we used C. dubliniensis as a model to investigate the role of TLO genes in regulating virulence and also to determine whether TLO paralogs have evolved to regulate distinct functions. A C. dubliniensis tlo1Δ/tlo2Δ mutant is unable to form true hyphae, has longer doubling times in galactose broth, is more susceptible to oxidative stress and forms increased levels of biofilm. Transcript profiling of the tlo1Δ/tlo2Δ mutant revealed increased expression of starvation responses in rich medium and retarded expression of hypha-induced transcripts in serum. ChIP studies indicated that Tlo1 binds to many ORFs including genes that exhibit high and low expression levels under the conditions analyzed. The altered expression of these genes in the tlo1Δ/tlo2Δ null mutant indicates roles for Tlo proteins in transcriptional activation and repression. Complementation of the tlo1Δ/tlo2Δ mutant with TLO1, but not TLO2, restored wild-type filamentous growth, whereas only TLO2 fully suppressed biofilm growth. Complementation with TLO1 also had a greater effect on doubling times in galactose broth. The different abilities of TLO1 and TLO2 to restore wild-type functions was supported by transcript profiling studies that showed that only TLO1 restored expression of hypha-specific genes (UME6, SOD5) and galactose utilisation genes (GAL1 and GAL10), whereas TLO2 restored repression of starvation-induced gene transcription. Thus, Tlo/Med2 paralogs encoding Mediator subunits regulate different virulence properties in Candida spp. and their expansion may account for the increased adaptability of C. albicans relative to other Candida species., Author Summary Candida albicans and C. dubliniensis are fungal pathogens of humans. Both species possess TLO genes encoding proteins with homology to the Med2 subunit of Mediator. The more virulent pathogen C. albicans has 15 copies of the TLO gene whereas the less pathogenic species C. dubliniensis has only two (TLO1 and TLO2). In this study we show that a C. dubliniensis mutant missing both TLO1 and TLO2 is defective in virulence functions, including hyphal growth and stress responses but forms increased levels of biofilm. Analysis of gene expression in the tlo1Δ/tlo2Δ mutant revealed extensive differences relative to wild-type cells, including aberrant expression of starvation responses in nutrient-rich medium and retarded expression of hypha-induced transcripts in serum. Tlo1 protein was found to interact with genes and this was associated with both gene activation and repression. TLO1 was found to be better at restoring hyphal growth compared to TLO2 and but was less effective than TLO2 in supressing biofilm formation in the tlo1Δ/tlo2Δ strain. Thus, Tlo proteins regulate many virulence properties in Candida spp. and the expansion of the TLO family in C. albicans may account for the increased adaptability of this species relative to other Candida species.

Mediator, a multiprotein complex involved in the regulation of RNA polymerase II transcription, binds to nucleosomes and acetylates histones. Three lines of evidence identify the Nut1 subunit of Mediator as responsible for the histone acetyltransferase (HAT) activity. An "in-gel" HAT assay reveals a single band of the appropriate size. Sequence alignment shows significant similarity of Nut1 to the GCN5-related N-acetyltransferase superfamily. Finally, recombinant Nut1 exhibits HAT activity in an in-gel assay.

Deletion of any one of three subunits of the yeast Mediator of transcriptional regulation, Med2, Pgd1 (Hrs1), and Sin4, abolished activation by Gal4–VP16 in vitro . By contrast, other Mediator functions, stimulation of basal transcription and of TFIIH kinase activity, were unaffected. A different but overlapping Mediator subunit dependence was found for activation by Gcn4. The genetic requirements for activation in vivo were closely coincident with those in vitro . A whole genome expression profile of a Δmed2 strain showed diminished transcription of a subset of inducible genes but only minor effects on “basal” transcription. These findings make an important connection between transcriptional activation in vitro and in vivo , and identify Mediator as a “global” transcriptional coactivator.

Protocols are presented for the preparation of a fully defined yeast RNA polymerase II transcription system, consisting of essentially pure TFIIB, -E, -F, and -H, TATA-binding protein, RNA polymerase II, and mediator of transcriptional regulation. This system, comprising 44 polypeptides, is able to initiate transcription at any of a dozen yeast and mammalian promoters thus far tested and responds to a variety of transcriptional activator proteins.

The amplification of the TLO (for t e lo mere-associated) genes in Candida albicans , compared to its less pathogenic, close relative Candida dubliniensis , suggests a role in virulence. Little, however, is known about the function of the Tlo proteins. We have purified the Mediator coactivator complex from C. albicans (caMediator) and found that Tlo proteins are a stoichiometric component of caMediator. Many members of the Tlo family are expressed, and each is a unique member of caMediator. Protein expression analysis of individual Tlo proteins, as well as the purification of tagged Tlo proteins, demonstrate that there is a large free population of Tlo proteins in addition to the Mediator-associated population. Coexpression and copurification of Tloα12 and caMed3 in Escherichia coli established a direct physical interaction between the two proteins. We have also made a C. albicans med3Δ/Δ strain and purified an intact Mediator from this strain. The analysis of the composition of the med3Δ Mediator shows that it lacks a Tlo subunit. Regarding Mediator function, the med3Δ/Δ strain serves as a substitute for the difficult-to-make tloΔ/Δ C. albicans strain. A potential role of the TLO and MED3 genes in virulence is supported by the inability of the med3Δ/Δ strain to form normal germ tubes. This study of caMediator structure provides initial clues to the mechanism of action of the Tlo genes and a platform for further mechanistic studies of caMediator's involvement in gene regulatory patterns that underlie pathogenesis.

DNA repair mechanisms maintain the integrity of genetic information. Recent discoveries have provided evidence for interplay between DNA repair and cell development and signaling. In the past year, atomic resolution studies of several proteins that participate in the repair of oxidative and alkylation damage, in addition to a variety of biochemical and biophysical studies, have provided much insight into the molecular mechanisms underlying DNA repair.

The Mediator complex is required for the regulated transcription of nearly all RNA polymerase II-dependent genes. Here we demonstrate a new role for Mediator which appears to be separate from its function as a transcriptional coactivator. Mediator associates directly with heterochromatin at telomeres and influences the exact boundary between active and inactive chromatin. Loss of the Mediator Med5 subunit or mutations in Med7 cause a depletion of the complex from regions located near subtelomeric X elements, which leads to a change in the balance between the Sir2 and Sas2 proteins. These changes in turn result in increased levels of H4K16 acetylation near telomeres and in desilencing of subtelomeric genes. Increases in H4K16 acetylation have been observed at telomeres in aging cells. In agreement with this observation, we found that the loss of MED5 leads to shortening of the Saccharomyces cerevisiae (budding yeast) replicative life span.

The Escherichia coli Ada protein repairs O6-methylguanine residues and methyl phosphotriesters in DNA by direct transfer of the methyl group to a cysteine residue located in its C- or N-terminal domain, respectively. Methyl transfer to the N-terminal domain causes it to acquire a sequence-specific DNA binding activity, which directs binding to the regulatory region of several methylation-resistance genes. In this paper we show that the N-terminal domain of Ada contains a high-affinity binding site for a single zinc atom, whereas the C-terminal domain is free of zinc. The metal-binding domain is apparently located within the first 92 amino acids of Ada, which contains four conserved cysteine residues. We propose that these four cysteines serve as the zinc ligand residues, coordinating the metal in a tetrahedral arrangement. One of the putative ligand residues, namely, Cys69, also serves as the acceptor site for a phosphotriester-derived methyl group. This raises the possibility that methylation-dependent ligand reorganization about the metal plays a role in the conformational switching mechanism that converts Ada from a non-sequence-specific to a sequence-specific DNA-binding protein.

The Saccharomyces cerevisiae Mediator is a 25-subunit complex that facilitates both transcriptional activation and repression. Structural and functional studies have divided Mediator subunits into four distinct modules. The Head, Middle, and Tail modules form the core functional Mediator complex, whereas a fourth, the Cyc-C module, is variably associated with the core. By purifying Mediator from a strain lacking the Med19(Rox3) subunit, we have found that a complex missing only the Med19(Rox3) subunit can be isolated under mild conditions. Additionally, we have established that the entire Middle module is released when the Deltamed19(rox3) Mediator is purified under more stringent conditions. In contrast to most models of the modular structure of Mediator, we show that release of the Middle module in the Deltamed19(rox3) Mediator leaves a stable complex made up solely of Head and Tail subunits. Both the intact and Head-Tail Deltamed19(rox3) Mediator complexes have defects in enhanced basal transcription, enhanced TFIIH phosphorylation of the CTD, as well as binding of RNA Pol II and the CTD. The largely intact Deltamed19(rox3) complex facilitates activated transcription at levels similar to the wild type Mediator. In the absence of the Middle module, however, the Deltamed19(rox3) Mediator is unable to facilitate activated transcription. Although the Middle module is unnecessary for holding the Head and Tail modules together, it is required for the complex to function as a conduit between activators and the core transcription machinery.

Med5 (Nut1) is identified here as a component of the Mediator tail region. Med5 is positioned peripherally to Med16 (Sin4) together with the three members of the putative Gal11 module, Med15 (Gal11), Med2, and Med3 (Pgd1). The biochemical analysis receives support from genetic interactions between med5delta and med15delta deletions. The med5delta and med16delta deletion strains share many phenotypes, including effects on mitochondrial function with enhanced growth on nonfermentable carbon sources, increased citrate synthase activity, and increased oxygen consumption. Deletion of the MED5 gene leads to increased transcription of nuclear genes encoding components of the oxidative phosphorylation machinery, whereas mitochondrial genes encoding components of the same machinery are down-regulated. We discuss a possible role for Med5 in coordinating nuclear and mitochondrial gene transcription.

The basal transcription factor TFIID is composed of the TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). Although TBP alone binds to the TATA box of DNA and supports basal transcription, the TAFs have essential functions that remain poorly defined. In order to study its properties, TFIID was purified from Saccharomyces cerevisiae using a newly developed affinity tag. Analysis of the final elution by mass spectrometry confirms the presence of all the known TAFs and TBP, as well as Rsp5, Bul1, Ubp3, Bre5, Cka1, and Cka2. Both Taf1 and Taf5 are ubiquitinated, and the ubiquitination pattern of TFIID changes when BUL1 or BRE5 is deleted. Purified TFIID binds specifically to promoter DNA in a manner stabilized by TFIIA, and these complexes can be analyzed by native gel electrophoresis. Phenanthroline-copper footprinting and photoaffinity cross-linking indicate that TFIID makes extensive contacts upstream and downstream of the TATA box. TFIID supports basal transcription and activated transcription, both of which are enhanced by TFIIA.

In Saccharomyces cerevisiae, Kin28 is a member of the cyclin-dependent kinase family. Kin28 is a subunit of the basal transcription factor holo-TFIIH and its trimeric sub-complex TFIIK. Kin28 is the primary kinase that phosphorylates the RNA polymerase II (RNA pol II) C-terminal domain (CTD) within a transcription initiation complex. Mediator, a global transcriptional co-activator, dramatically enhances the phosphorylation of the CTD of RNA pol II by holo-TFIIH in vitro. Using purified proteins we have determined that the subunits of TFIIK are sufficient for Mediator to enhance Kin28 CTD kinase activity and that Mediator enhances phosphorylation of a glutathione S-transferase-CTD fusion protein, despite the absence of multiple Mediator and/or TFIIH interactions with polymerase. Mediator does not stimulate the activity of several other CTD kinases, suggesting that the specific enhancement of TFIIH kinase activity results in Kin28 being the primary CTD kinase at initiation. In addition, we have found that Kin28 phosphorylates Mediator subunit Med4 in an assay, including purified holo-TFIIH, and either Mediator or recombinant Med4 alone. Furthermore, Kin28 appears to be, at least in part, responsible for the phosphorylation of Med4 in vivo. We have identified Thr-237 as the site of phosphorylation of Med4 by Kin28 in vitro. The mutation of Thr-237 to Ala has no effect on the growth of a yeast strain under normal conditions but confirms that Thr-237 is also the site of Med4 phosphorylation in vivo.

Using a highly pure transcription system derived from Saccharomyces cerevisiae, we have purified an activity in yeast whole-cell extracts that represses RNA polymerase II transcription. Mechanistic studies suggest that this repressor specifically targets transcriptional reinitiation. The two polypeptides that constitute the repressor have been identified as Ceg1p and Cet1p, the two subunits of the yeast pre-mRNA capping enzyme. A purified recombinant capping enzyme is able to reconstitute repressor activity. Cet1p is necessary for and capable of this repression. Transcriptional run-on experiments indicate that the capping enzyme also serves as a repressor in vivo. Efficient pre-mRNA capping relies on interactions between the capping enzyme and transcription apparatus. Repression by the capping enzyme suggests a bidirectional flow of information between capping and transcription.

Candida albicans biofilm formation is a key virulence trait that involves hyphal growth and adhesin expression. Pyocyanin (PYO), a phenazine secreted by Pseudomonas aeruginosa, inhibits both C. albicans biofilm formation and development of wrinkled colonies. Using a genetic screen, we identified two mutants, ssn3Δ/Δ and ssn8Δ/Δ, which continued to wrinkle in the presence of PYO. Ssn8 is a cyclin-like protein and Ssn3 is similar to cyclin-dependent kinases; both proteins are part of the heterotetrameric Cdk8 module that forms a complex with the transcriptional co-regulator, Mediator. Ssn3 kinase activity was also required for PYO sensitivity as a kinase dead mutant maintained a wrinkled colony morphology in the presence of PYO. Furthermore, similar phenotypes were observed in mutants lacking the other two components of the Cdk8 module—Srb8 and Srb9. Through metabolomics analyses and biochemical assays, we showed that a compromised Cdk8 module led to increases in glucose consumption, glycolysis-related transcripts, oxidative metabolism and ATP levels even in the presence of PYO. In the mutant, inhibition of respiration to levels comparable to the PYO-treated wild type inhibited wrinkled colony development. Several lines of evidence suggest that PYO does not act through Cdk8. Lastly, the ssn3 mutant was a hyperbiofilm former, and maintained higher biofilm formation in the presence of PYO than the wild type. Together these data provide novel insights into the role of the Cdk8 module of Mediator in regulation of C. albicans physiology and the links between respiratory activity and both wrinkled colony and biofilm development., Author Summary Candida albicans is currently one of the most common causes of nosocomial infections, and causes diseases ranging from oral thrush to life-threatening systemic infections. C. albicans readily forms biofilms on implanted devices, such as catheters and dentures, and biofilms are associated with increased risk of systemic infections and resistance to antifungals. We previously showed that biofilm formation positively correlates with oxidative metabolism. Here, we show that respiration, wrinkled colony formation, biofilm formation, and resistance to the inhibitory effects of pyocyanin were increased when the Cdk8 module of Mediator, a co-regulator of transcription, was compromised. Cdk8 module mutants all exhibited differences in basal metabolism that were indicative of increased metabolic activity. Furthermore, using these strains, we showed a direct correlation between the inhibition of respiratory activity, in the absence of growth defects, and inhibition of biofilm formation, supporting a model in which C. albicans metabolism and morphology are linked. The human Cdk8 module is currently being investigated as a target for cancer chemotherapy, and thus an understanding of the consequences or potential benefits of inhibiting this module is required.

Structures of yeast Mediator complex, of a related complex from mouse cells and of thyroid hormone receptor-associated protein complex from human cells have been determined by three-dimensional reconstruction from electron micrographs of single particles. All three complexes show a division in two parts, a “head” domain and a combined “middle-tail” domain. The head domains of the three complexes appear most similar and interact most closely with RNA polymerase II. The middle-tail domains show the greatest structural divergence and, in the case of the tail domain, may not interact with polymerase at all. Consistent with this structural divergence, analysis of a yeast Mediator mutant localizes subunits that are not conserved between yeast and mammalian cells to the tail domain. Biochemically defined Rgr1 and Srb4 modules of yeast Mediator are then assigned to the middle and head domains.

▪ Abstract Three lines of evidence have converged on a multiprotein Mediator complex as a conserved interface between gene-specific regulatory proteins and the general transcription apparatus of eukaryotes. Mediator was discovered as an activity required for transcriptional activation in a reconstituted system from yeast. Upon resolution to homogeneity, the activity proved to reside in a 20-protein complex, which could exist in a free state or in a complex with RNA polymerase II, termed holoenzyme. A second line of evidence came from screens in yeast for mutations affecting transcription. Two-thirds of Mediator subunits are encoded by genes revealed by these screens. Five of the genetically defined subunits, termed Srbs, were characterized as interacting with the C-terminal domain of RNA polymerase II in vivo, and were shown to bind polymerase in vitro. A third line of evidence has come recently from studies in mammalian transcription systems. Mammalian counterparts of yeast Mediator were shown to interact with transcriptional activator proteins and to play an essential role in transcriptional regulation. Mediator evidently integrates and transduces positive and negative regulatory information from enhancers and operators to promoters. It functions directly through RNA polymerase II, modulating its activity in promoter-dependent transcription. Details of the Mediator mechanism remain obscure. Additional outstanding questions include the patterns of promoter-specificity of the various Mediator subunits, the possible cell-type–specificity of Mediator subunit composition, and the full structures of both free Mediator and RNA polymerase II holoenzyme.

Single particles of the mediator of transcriptional regulation (Mediator) and of RNA polymerase II holoenzyme were revealed by electron microscopy and image processing. Mediator alone appeared compact, but at high pH or in the presence of RNA polymerase II it displayed an extended conformation. Holoenzyme contained Mediator in a fully extended state, partially enveloping the globular polymerase, with points of apparent contact in the vicinity of the polymerase carboxyl-terminal domain and the DNA-binding channel. A similarity in appearance and conformational behavior of yeast and murine complexes indicates a conservation of Mediator structure among eukaryotes.

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.

The 10kDa amino-terminal fragment of Escherichia coli Ada protein (N-Ada10) repairs methyl phosphotriesters in DNA and possesses a tightly bound zinc ion. The complete resonance assignments of this protein domain have been obtained using multidimensional homonuclear and heteronuclear NMR experiments. The assignments served to study the internal mobility of this protein domain via 15N relaxation experiments. This involved the measurement of longitudinal and transverse 15N relaxation rates, as well as the amide proton solvent exchange rates. Relaxation rates in the rotating frame, R1 rho, of 15N nuclei were measured at different spin-lock field strengths, leading to the detection of two slow conformational exchange processes at Gly-25 and Gln-73. For the latter, which is next to the active site of this protein domain, the characteristic time of this process was found to be around 60 microseconds. The other relaxation experiments unveiled some regions of fast internal motions, faster than the overall correlation time. These motions were found in the N- and C- terminal tails, in segment 33-35 which forms the turn between beta-strands S1 and S2, and residues 47-52 located in a long loop preceding strand S3. The latter loop belongs to the potential DNA binding surface of N-Ada10. While the structure from residue 18 to residue 26 appears not well defined in the calculated structure, the relaxation experiments do not indicate higher mobility for this region. Residues at the N-terminal portion, including the first helix, the sequentially adjacent loop, and part of the second helix, exhibit internal motions close to the time scale of the overall rotational correlation time. This appears to be related to the fact that the first helix has no hydrogen bonds or salt bridges to the rest of the protein and is stabilized only by the involvement of some of its side chains in a hydrophobic core consisting of the side chains of two phenylalanines, a tryptophan, a leucine, and a valine. The four cysteines which bind the zinc show motions on different time scales ranging from microseconds to picoseconds. Thus the motions in the immediate region around the bound zinc of the DNA methyl phosphotriester repair domain are of relatively small amplitude but take place over a wide time range. On the other hand, high mobility is found in the turn connecting S1 and S2 and in the loop preceding S3, regions of the potential DNA binding surface.

The Escherichia coli Ada protein repairs methylphosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteine residues. The methyl transfer process is autocatalyzed by coordination of the acceptor residue, Cys69, to a tightly bound zinc ion. Kinetic data reveal a 4-fold reduction in the methylphosphotriester repair activity for the Cd(II) form of Ada versus the native Zn(II)-bound form, thus confirming a direct role for the metal in autocatalysis. Quantitative electrophoretic mobility shift assays reveal that the specific DNA affinity of the protein is increased 10(3)-fold by transfer of a methyl group to Cys69; the Cd(II) and the Zn(II) forms of the protein behave similarly in this respect. This methylation-sensitive stimulation of binding underlies the ability of Ada to activate inducibly the transcription of a methylation-dependent regulon. We conclude that the chemical properties of the bound metal influence the transition state for autocatalytic methyl transfer, but not the structure that ultimately results from this process.

Backgroud: The Ada protein of Escherichia coli repairs methyl phosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteine residues. This residue, Cys69, is ligated to a tightly bound zinc ion in the protein. After methyl transfer, Ada can bind DNA sequence-specifically, inducing the transcription of genes that confer resistance to the toxic effects of methylating agents. Coordination of zinc via a thioether-S is exceedingly rare. We therefore investigated whether methylation causes ligand exchange of Cys69, replacing the thioether with a new zinc ligand with higher affinity for the metal. Results: We added a 13 C-labeled methyl group to Cys69 of Ada and used isotope-edited NMR to observe the behavior of its protons. Comparison of the spectra for the Zn- and 112 Cd- bound forms of the methylated protein with that of the 113 Cd-bound form provided clear evidence that S-Me-Cys69 is coordinated to the metal in Ada when Ada is bound specifically to DNA. Conclusions: The transcriptionally competent form of Ada, in which Cys69 is methylated and the protein is bound to DNA, maintains the coordination of S-MeCys69 to the metal ion. Thus, ligand exchange is not responsible for switching Ada from a DNA-repair protein to a transcriptional activator. We propose that the lability of the thioether-zinc coordinate bond may provide a mechanism for down-regulation of the adaptive response by inactivation of the Ada DNA-binding domain.

The Escherichia coli Ada protein repairs methyl phosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteine residues. The methyl-transfer process appears to be autocatalyzed by coordination of the acceptor residue, Cys-69, to a tightly bound zinc ion. Upon methyl transfer, Ada acquires the ability to bind DNA sequence-specifically and thereby to induce genes that confer resistance to methylating agents. The solution structure of an N-terminal 10-kDa fragment of Ada, which retains zinc binding and DNA methyl phosphotriester repair activities, was determined using multidimensional heteronuclear nuclear magnetic resonance techniques. The structure reveals a zinc-binding motif unlike any observed thus far in transcription factors or zinc-containing enzymes and provides insight into the mechanism of metalloactivated DNA repair.

The Escherichia coli Ada protein repairs methylphosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteines. Upon methyl transfer, Ada acquires the ability to bind specific DNA sequences and thereby to induce genes that confer resistance to methylating agents. The amino-terminal domain of Ada, which comprises the methylphosphotriester repair and sequence-specific DNA binding elements, contains a tightly bound zinc ion. Analysis of the zinc binding site by cadmium-113 nuclear magnetic resonance and site-directed mutagenesis revealed that zinc participates in the autocatalytic activation of the active site cysteine and may also function as a conformational switch.

When a eukaryotic cell divides, tension builds at centromeres as spindle forces pull chromosomes toward opposite poles during metaphase. New data show that centromeric chromatin stretches in response to these forces, revealing a mechanical role for chromatin packaging in mitosis.