A Gal4-sigma 54 hybrid protein that functions as a potent activator of RNA polymerase II transcription in yeast.

The bacterial final sigma(54) protein associates with core RNA polymerase to form a holoenzyme complex that renders cognate promoters enhancer-dependent. Although unusual in bacteria, enhancer-dependent transcription is the paradigm in eukaryotes. Here we report that a fragment of Escherichia coli final sigma(54) encompassing amino acid residues 29-177 functions as a potent transcriptional activator in yeast when fused to a Gal4 DNA binding domain. Activation by Gal4-final sigma(54) is TATA-dependent and requires the SAGA coactivator complex, suggesting that Gal4-final sigma(54) functions by a normal mechanism of transcriptional activation. Surprisingly, deletion of the AHC1 gene, which encodes a polypeptide unique to the ADA coactivator complex, stimulates Gal4-final sigma(54)-mediated activation and enhances the toxicity of Gal4-final sigma(54). Accordingly, the SAGA and ADA complexes, both of which include Gcn5 as their histone acetyltransferase subunit, exert opposite effects on transcriptional activation by Gal4-final sigma(54). Gal4-final sigma(54) activation and toxicity are also dependent upon specific final sigma(54) residues that are required for activator-responsive promoter melting by final sigma(54) in bacteria, implying that activation is a consequence of final sigma(54)-specific features rather than a structurally fortuitous polypeptide fragment. As such, Gal4-final sigma(54) represents a novel tool with the potential to provide insight into the mechanism by which natural activators function in eukaryotic cells.

Functional interaction between Ssu72 and the Rpb2 subunit of RNA polymerase II in Saccharomyces cerevisiae.

SSU72 is an essential gene encoding a phylogenetically conserved protein of unknown function that interacts with the general transcription factor TFIIB. A recessive ssu72-1 allele was identified as a synthetic enhancer of a TFIIB (sua7-1) defect, resulting in a heat-sensitive (Ts(-)) phenotype and a dramatic downstream shift in transcription start site selection. Here we describe a new allele, ssu72-2, that confers a Ts(-) phenotype in a SUA7 wild-type background. In an effort to further define Ssu72, we isolated suppressors of the ssu72-2 mutation. One suppressor is allelic to RPB2, the gene encoding the second-largest subunit of RNA polymerase II (RNAP II). Sequence analysis of the rpb2-100 suppressor defined a cysteine replacement of the phylogenetically invariant arginine residue at position 512 (R512C), located within homology block D of Rpb2. The ssu72-2 and rpb2-100 mutations adversely affected noninduced gene expression, with no apparent effects on activated transcription in vivo. Although isolated as a suppressor of the ssu72-2 Ts(-) defect, rpb2-100 enhanced the transcriptional defects associated with ssu72-2. The Ssu72 protein interacts directly with purified RNAP II in a coimmunoprecipitation assay, suggesting that the genetic interactions between ssu72-2 and rpb2-100 are a consequence of physical interactions. These results define Ssu72 as a highly conserved factor that physically and functionally interacts with the RNAP II core machinery during transcription initiation.

Genetic analysis of the YDR1-BUR6 repressor complex reveals an intricate balance among transcriptional regulatory proteins in yeast.

A transcriptional repressor complex encoded by two essential genes, YDR1 and BUR6, was isolated from Saccharomyces cerevisiae and shown to be the functional counterpart of the human repressor complex Dr1-DRAP1. To elucidate the mechanism of repression by this complex, altered forms of Ydr1 and Bur6 were studied in vitro and in vivo. Deletion of the C-terminal 41 amino acids of Ydr1 resulted in loss of repressor activity and a growth defect, suggesting that the C-terminal domain of Ydr1 functions as a potent transcriptional repressor. A screen for extragenic suppressors of a cold-sensitive ydr1 (ydr1(cs)) mutant led to the identification of recessive mutations in the SIN4 gene, which encodes a component of the SRB-MED complex. The sin4 alleles suppressed not only ydr1(cs) mutations but also bur6(cs) mutations. In contrast, deletion of the gal11 gene, whose product is also a member of the SRB-MED complex, failed to suppress ydr1(cs) and bur6(cs) mutations, indicating that suppression is not due to general defects in the SRB-MED complex. Moreover, one of the sin4 alleles, but not the sin4 deletion, was found to specifically suppress the inviability of a ydr1 deletion, demonstrating that the essential function of Ydr1 becomes dispensable in a sin4 mutant background. Biochemical analysis of the SRB-MED complex from the sin4 suppressor strain revealed a structurally distinct form of the SRB-MED complex that lacks a subset of mediator subunits. These results define a delicate balance between positive and negative regulators of transcription operating through the Ydr1-Bur6 repressor complex.

Mutational analysis of yeast TFIIB. A functional relationship between Ssu72 and Sub1/Tsp1 defined by allele-specific interactions with TFIIB.

TFIIB is an essential component of the RNA polymerase II core transcriptional machinery. Previous studies have defined TFIIB domains required for interaction with other transcription factors and for basal transcription in vitro. In the study reported here we investigated the TFIIB structural requirements for transcription initiation in vivo. A library of sua7 mutations encoding altered forms of yeast TFIIB was generated by error-prone polymerase chain reaction and screened for conditional growth defects. Twenty-two single amino acid replacements in TFIIB were defined and characterized. These replacements are distributed throughout the protein and occur primarily at phylogenetically conserved positions. Most replacements have little or no effect on the steady-state protein levels, implying that each affects TFIIB function rather than synthesis or stability. In contrast to the initial sua7 mutants, all replacements, with one exception, have no effect on start site selection, indicating that specific TFIIB structural defects affect transcriptional accuracy. This collection of sua7 alleles, including the initial sua7 alleles, was used to investigate the allele specificity of interactions between ssu72 and sub1, both of which were initially identified as either suppressors (SUB1 2mu) or enhancers (sub1Delta, ssu72-1) of sua7 mutations. We show that the interactions of ssu72-1 and sub1Delta with sua7 are allele specific; that the allele specificities of ssu72 and sub1 overlap; and that each of the sua7 alleles that interacts with ssu72 and sub1 affects the accuracy of transcription start site selection. These results demonstrate functional interactions among TFIIB, Ssu72, and Sub1 and suggest that these interactions play a role in the mechanism of start site selection by RNA polymerase II.

Transcription: Common cofactors and cooperative recruitment.

Mammalian counterparts of the yeast SRB/MED transcriptional 'mediator' complex have recently been identified. These complexes define a common cofactor requirement for diverse transcriptional activators and underscore the conserved nature of the transcriptional machinery among eukaryotic organisms.

The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins.

The regulation of gene expression depends critically upon chromatin structure. Transcription of protein-coding genes can be reconstituted on naked DNA with only the general transcription factors and RNA polymerase II. This minimal system cannot transcribe DNA packaged into chromatin, indicating that accessory factors may facilitate access to DNA. Two classes of accessory factor, ATP-dependent chromatin-remodelling enzymes and histone acetyltransferases, facilitate transcription initiation from chromatin templates. FACT (for facilitates chromatin transcription) is a chromatin-specific elongation factor required for transcription of chromatin templates in vitro. Here we show that FACT comprises a new human homologue of the Saccharomyces cerevisiae Spt16/Cdc68 protein and the high-mobility group-1-like protein structure-specific recognition protein-1. Yeast SPT16/CDC68 is an essential gene that has been implicated in transcription and cell-cycle regulation. Consistent with our biochemical analysis of FACT, we provide evidence that Spt16/Cdc68 is involved in transcript elongation in vivo. Moreover, FACT specifically interacts with nucleosomes and histone H2A/H2B dimers, indicating that it may work by promoting nucleosome disassembly upon transcription. In support of this model, we show that FACT activity is abrogated by covalently crosslinking nucleosomal histones.

A general requirement for the Sin3-Rpd3 histone deacetylase complex in regulating silencing in Saccharomyces cerevisiae.

The Sin3-Rpd3 histone deacetylase complex, conserved between human and yeast, represses transcription when targeted by promoter-specific transcription factors. SIN3 and RPD3 also affect transcriptional silencing at the HM mating loci and at telomeres in yeast. Interestingly, however, deletion of the SIN3 and RPD3 genes enhances silencing, implying that the Sin3-Rpd3 complex functions to counteract, rather than to establish or maintain, silencing. Here we demonstrate that Sin3, Rpd3, and Sap30, a novel component of the Sin3-Rpd3 complex, affect silencing not only at the HMR and telomeric loci, but also at the rDNA locus. The effects on silencing at all three loci are dependent upon the histone deacetylase activity of Rpd3. Enhanced silencing associated with sin3Delta, rpd3Delta, and sap30Delta is differentially dependent upon Sir2 and Sir4 at the telomeric and rDNA loci and is also dependent upon the ubiquitin-conjugating enzyme Rad6 (Ubc2). We also show that the Cac3 subunit of the CAF-I chromatin assembly factor and Sin3-Rpd3 exert antagonistic effects on silencing. Strikingly, deletion of GCN5, which encodes a histone acetyltransferase, enhances silencing in a manner similar to deletion of RPD3. A model that integrates the effects of rpd3Delta, gcn5Delta, and cac3Delta on silencing is proposed.

RNA polymerase II as a control panel for multiple coactivator complexes.

1999 marks the 30th anniversary of the reported discovery of sigma factor and the bacterial RNA polymerase holoenzyme. In 1994, an RNA polymerase II complex was discovered in yeast - mammalian complexes were subsequently identified. Recent developments regarding the composition and function of RNA polymerase II complexes suggest, however, that the concept of the holoenzyme, as defined in bacteria, might not be relevant to eukaryotes.

An activation-specific role for transcription factor TFIIB in vivo.

A yeast mutant was isolated encoding a single amino acid substitution [serine-53 --> proline (S53P)] in transcription factor TFIIB that impairs activation of the PHO5 gene in response to phosphate starvation. This effect is activation-specific because S53P did not affect the uninduced level of PHO5 expression, yet is not specific to PHO5 because Adr1-mediated activation of the ADH2 gene also was impaired by S53P. Pho4, the principal activator of PHO5, directly interacted with TFIIB in vitro, and this interaction was impaired by the S53P replacement. Furthermore, Pho4 induced a conformational change in TFIIB, detected by enhanced sensitivity to V8 protease. The S53P replacement also impaired activation of a lexA(op)-lacZ reporter by a LexA fusion protein to the activation domain of Adr1, thereby indicating that the transcriptional effect on ADH2 expression is specific to the activation function of Adr1. These results define an activation-specific role for TFIIB in vivo and suggest that certain activators induce a conformational change in TFIIB as part of their mechanism of transcriptional stimulation.

SAP30, a novel protein conserved between human and yeast, is a component of a histone deacetylase complex.

Histone acetylation plays a key role in the regulation of eukaryotic gene expression. Recently, histone acetylation and deacetylation were found to be catalyzed by structurally distinct, multisubunit complexes that mediate, respectively, activation and repression of transcription. Here, we identify SAP30 as a novel component of the human histone deacetylase complex that includes Sin3, the histone deacetylases HDAC1 and HDAC2, histone binding proteins RbAp46 and RbAp48, as well as other polypeptides. Moreover, we describe a SAP30 homolog in yeast that is functionally related to Sin3 and the histone deacetylase Rpd3. The human SAP30 complex is active in deacetylating core histone octamers, but inactive in deacetylating nucleosomal histones due to the inability of the histone binding proteins RbAp46 and RbAp48 to gain access to nucleosomal histones. These results define SAP30 as a component of a histone deacetylase complex conserved among eukaryotic organisms.

The HIS4 gene from the yeast Kluyveromyces lactis.

The Kluyveromyces lactis HIS4 gene was cloned by complementation of a Saccharomyces cerevisiae his4 mutant. Sequence analysis revealed a 2388 bp open reading frame encoding a single polypeptide predicted to encompass three distinct enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase). This structural organization is strikingly similar to that of the His4 proteins from S. cerevisiae and Pichia pastoris. Transcript analysis detected a single mRNA species of 2.5 kb.

Molecular genetics of the RNA polymerase II general transcriptional machinery.

Transcription initiation by RNA polymerase II (RNA pol II) requires interaction between cis-acting promoter elements and trans-acting factors. The eukaryotic promoter consists of core elements, which include the TATA box and other DNA sequences that define transcription start sites, and regulatory elements, which either enhance or repress transcription in a gene-specific manner. The core promoter is the site for assembly of the transcription preinitiation complex, which includes RNA pol II and the general transcription fctors TBP, TFIIB, TFIIE, TFIIF, and TFIIH. Regulatory elements bind gene-specific factors, which affect the rate of transcription by interacting, either directly or indirectly, with components of the general transcriptional machinery. A third class of transcription factors, termed coactivators, is not required for basal transcription in vitro but often mediates activation by a broad spectrum of activators. Accordingly, coactivators are neither gene-specific nor general transcription factors, although gene-specific coactivators have been described in metazoan systems. Transcriptional repressors include both gene-specific and general factors. Similar to coactivators, general transcriptional repressors affect the expression of a broad spectrum of genes yet do not repress all genes. General repressors either act through the core transcriptional machinery or are histone related and presumably affect chromatin function. This review focuses on the global effectors of RNA polymerase II transcription in yeast, including the general transcription factors, the coactivators, and the general repressors. Emphasis is placed on the role that yeast genetics has played in identifying these factors and their associated functions.

A review of phenotypes in Saccharomyces cerevisiae.

A summary of previously defined phenotypes in the yeast Saccharomyces cerevisiae is presented. The purpose of this review is to provide a compendium of phenotypes that can be readily screened to identify pleiotropic phenotypes associated with primary or suppressor mutations. Many of these phenotypes provide a convenient alternative to the primary phenotype for following a gene, or as a marker for cloning a gene by genetic complementation. In many cases a particular phenotype or set of phenotypes can suggest a function for the product of the mutated gene.

The Dr1/DRAP1 heterodimer is a global repressor of transcription in vivo.

A general repressor extensively studied in vitro is the human Dr1/DRAP1 heterodimeric complex. To elucidate the function of Dr1 and DRAP1 in vivo, the yeast Saccharomyces cerevisiae Dr1/DRAP1 repressor complex was identified. The repressor complex is encoded by two essential genes, designated YDR1 and BUR6. The inviability associated with deletion of the yeast genes can be overcome by expressing the human genes. However, the human corepressor DRAP1 functions in yeast only when human Dr1 is coexpressed. The yDr1/Bur6 complex represses transcription in vitro in a reconstituted RNA polymerase II transcription system. Repression of transcription could be overcome by increasing the concentration of TATA-element binding protein (TBP). Consistent with the in vitro results, overexpression of YDR1 in vivo resulted in decreased mRNA accumulation. Furthermore, YDR1 overexpression impaired cell growth, an effect that could be rescued by overexpression of TBP. In agreement with our previous studies in vitro, we found that overexpression of Dr1 in vivo also affected the accumulation of RNA polymerase III transcripts, but not of RNA polymerase I transcripts. Our results demonstrate that Dr1 functions as a repressor of transcription in vivo and, moreover, directly targets TBP, a global regulator of transcription.

Transcription: why are TAFs essential?

The TAFs are transcription factors associated with the TATA-binding protein, and until recently they were assumed to link specific activators to the general transcription machinery. Recent results suggest that the essential functions of TAFs are not as coactivators of transcription but as determinants of promoter selectivity.

Functional interaction between TFIIB and the Rpb9 (Ssu73) subunit of RNA polymerase II in Saccharomyces cerevisiae.

Recessive mutations in the SSU71, SSU72 and SSU73 genes of Saccharomyces cerevisiae were identified as either suppressors or enhancers of a TFIIB defect (sua7-1) that confers both a cold-sensitive growth phenotype and a downstream shift in transcription start site selection. The SSU71 (TFG1) gene encodes the largest subunit of TFIIF and SSU72 encodes a novel protein that is essential for cell viability. Here we report that SSU73 is identical to RPB9, the gene encoding the 14.2 kDa subunit of RNA polymerase II. The ssu73-1 suppressor compensates for both the growth defect and the downstream shift in start site selection associated with sua7-1. These effects are similar to those of the ssu71-1 suppressor and distinct from the ssu72-1 enhancer. The ssu73-1 allele was retrieved and sequenced, revealing a nonsense mutation at codon 107. Consequently, ssu73-1 encodes a truncated form of Rpb9 lacking the C-terminal 16 amino acids. This Rpb9 derivative retains at least partial function since the ssu73-1 mutant exhibits none of the growth defects associated with rpb9 null mutants. However, in a SUA7+ background, ssu73-1 confers the same upstream shift at ADH1 as an rpb9 null allele. This suggests that the C-terminus of Rpb9 functions in start site selection and demonstrates that the previously observed effects of rpb9 mutations on start site selection are not necessarily due to complete loss of function. These results establish a functional interaction between TFIIB and the Rpb9 subunit of RNA polymerase II and suggest that these two components of the preinitiation complex interact during transcription start site selection.