ABSTRACT
Histone acetyltransferases (HATs) such as Gcn5 play a role in transcriptional activation. However, the majority of constitutive genes show no requirement for GCN5, and even regulated genes, such as the yeast PHO5 gene, do not seem to be affected significantly by its absence under normal activation conditions. Here we show that even though the steady-state level of activated PHO5 transcription is not affected by deletion of GCN5, the rate of activation following phosphate starvation is significantly decreased. This delay in transcriptional activation is specifically due to slow chromatin remodeling of the PHO5 promoter, whereas the transmission of the phosphate starvation signal to the PHO5 promoter progresses at a normal rate. Chromatin remodeling is equally delayed in a galactose-inducible PHO5 promoter variant in which the Pho4 binding sites have been replaced by Gal4 binding sites. By contrast, activation of the GAL1 gene by galactose addition occurs with normal kinetics. Lack of the histone H4 N-termini leads to a similar delay in activation of the PHO5 promoter. These results indicate that one important contribution of HATs is to increase the rate of gene induction by accelerating chromatin remodeling, rather than to affect the final steady-state expression levels.
Subject(s)
Acetyltransferases/metabolism , Chromatin/physiology , DNA-Binding Proteins , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Membrane Transport Proteins/genetics , Phosphate Transport Proteins , Protein Kinases/genetics , Protein Kinases/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Acetyltransferases/genetics , Chromatin/genetics , Enzyme Induction , Gene Expression Regulation, Enzymologic , Genes, Reporter , Green Fluorescent Proteins , Histone Acetyltransferases , Histones/metabolism , Kinetics , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Membrane Transport Proteins/biosynthesis , Phosphates/metabolism , Promoter Regions, Genetic , Recombinant Fusion Proteins/analysis , Transcriptional ActivationABSTRACT
Elongator is a histone acetyltransferase complex that associates with the elongating form of RNA polymerase II. We purified Elongator to virtual homogeneity via a rapid three-step procedure based largely on affinity chromatography. The purified factor, holo-Elongator, is a labile six-subunit factor composed of two discrete subcomplexes: one comprised of the previously identified Elp1, Elp2, and Elp3 proteins and another comprised of three novel polypeptides, termed Elp4, Elp5, and Elp6. Disruption of the yeast genes encoding the new Elongator proteins confers phenotypes indistinguishable from those previously described for the other elp mutants, and concomitant disruption of genes encoding proteins in either subcomplex does not confer new phenotypes. Taken together, our results indicate that holo-Elongator is a functional entity in vitro as well as in vivo. Metazoan homologues of Elp1 and Elp3 have previously been reported. We cloned the human homologue of yeast ELP4 and show that this gene is ubiquitously expressed in human tissues.
Subject(s)
Acetyltransferases/chemistry , Acetyltransferases/metabolism , RNA Polymerase II/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Acetyltransferases/genetics , Acetyltransferases/isolation & purification , Amino Acid Sequence , Animals , Caenorhabditis elegans/genetics , Cloning, Molecular , Conserved Sequence , Drosophila melanogaster/genetics , Histone Acetyltransferases , Humans , Macromolecular Substances , Mice , Molecular Sequence Data , Peptide Fragments/chemistry , Phenotype , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Schizosaccharomyces/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Transcription, GeneticABSTRACT
Yeast cells lacking transcription elongation factor genes such as PPR2 (TFIIS) and ELP (Elongator) are viable and show deleterious phenotypes only when transcription is rendered less effective by RNA polymerase mutations or by decreasing nucleotide pools. Here we demonstrate that deletion of the CTK1 gene, encoding the kinase subunit of RNA polymerase II carboxy-terminal domain kinase I (CTDK-I), is synthetically lethal when combined with deletion of PPR2 or ELP genes. The inviability of ctk1 elp3 double mutants can be rescued by expression of an Elp3 mutant that has retained its ability to form the Elongator complex but has severely diminished histone acetyltransferase activity, suggesting that the functional overlap between CTDK-I and Elongator is in assembly of RNA polymerase II elongation complexes. Our results suggest that CTDK-I plays an important role in transcriptional elongation in vivo, possibly by creating a form of RNA polymerase that is less prone to transcriptional arrest.
Subject(s)
Protein Kinases/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Transcription Factors, General , Transcription, Genetic/genetics , Transcriptional Elongation Factors , Acetyltransferases/genetics , Cell Division/drug effects , Cell Division/genetics , Gene Deletion , Genes, Lethal , Histone Acetyltransferases , Mutation , Protein Kinases/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/growth & development , Transcription Factors/genetics , Uracil/analogs & derivatives , Uracil/pharmacologyABSTRACT
Transcription-induced recombination has been reported in all organisms from bacteria to mammals. We have shown previously that the yeast genes HPR1 and THO2 may be keys to the understanding of transcription-associated recombination, as they both affect transcription elongation and hyper-recombination in a concerted manner. Using a yeast strain that has the wild-type THO2 gene replaced by one encoding a His(6)-HA-tagged version, we have isolated an oligomeric complex containing four proteins: Tho2, Hpr1, Mft1 and a novel protein that we have named Thp2. We have reciprocally identified a complex containing Hpr1, Tho2 and Mft1 using anti-Mft1 antibodies in immunoprecipitation experiments. The protein complex is mainly nuclear; therefore, Tho2 and Hpr1 are physically associated. Like hpr1Delta and tho2Delta cells, mft1Delta and thp2Delta cells show mitotic hyper- recombination and impaired transcription elongation, in particular, through the bacterial lacZ sequence. Hyper-recombination conferred by mft1Delta and thp2Delta is only observed in DNA regions under transcription conditions. We propose that this protein complex acts as a functional unit connecting transcription elongation with the incidence of mitotic recombination.
Subject(s)
DNA-Binding Proteins , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Animals , Base Sequence , DNA Primers/genetics , Fungal Proteins/genetics , Gene Expression , Genes, Fungal , Macromolecular Substances , Mitosis , Mutation , Nuclear Proteins , Phenotype , Recombination, Genetic , Saccharomyces cerevisiae/cytology , Subcellular Fractions/metabolism , Temperature , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Elp3 and Gcn5 are histone acetyltransferases (HATs) that function in transcription as subunits of Elongator and SAGA/ADA, respectively. Here we show that mutations that impair the in vitro HAT activity of Elp3 confer typical elp phenotypes such as temperature sensitivity. Combining an elp3Delta mutation with histone H3 or H4 tail mutations confers lethality or sickness, supporting a role for Elongator in chromatin remodelling in vivo. gcn5Deltaelp3Delta double mutants display a number of severe phenotypes, and similar phenotypes result from combining the elp mutation with mutation in a gene encoding a SAGA-specific, but not an ADA-specific subunit, indicating that Elongator functionally overlaps with SAGA. Because concomitant active site alterations in Elp3 and Gcn5 are sufficient to confer severe phenotypes, the redundancy must be specifically related to the HAT activity of these complexes. In support of this conclusion, gcn5Deltaelp3Delta phenotypes are suppressed by concomitant mutation of the HDA1 and HOS2 histone deacetylases. Our results demonstrate functional redundancy among transcription-associated HAT and deacetylase activities, and indicate the importance of a fine-tuned acetylation-deacetylation balance during transcription in vivo.
Subject(s)
Acetyltransferases/metabolism , Chromatin/metabolism , DNA-Binding Proteins , Fungal Proteins/metabolism , Protein Kinases/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Acetylation , Acetyltransferases/genetics , Histone Acetyltransferases , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Mutation , Phenotype , Transcription Factors/metabolismABSTRACT
A novel yeast gene, ELP2, is shown to encode the 90-kDa subunit of the Elongator complex and elongating RNA polymerase II holoenzyme. ELP2 encodes a protein with eight WD40 repeats, and cells lacking the gene display typical elp phenotypes, such as temperature and salt sensitivity. Generally, different combinations of double and triple ELP gene deletions cause the same phenotypes as single ELP1, ELP2, or ELP3 deletion, providing genetic evidence that the ELP gene products work together in a complex.
Subject(s)
Holoenzymes/chemistry , RNA Polymerase II/chemistry , RNA Polymerase II/physiology , Amino Acid Sequence , Blotting, Western , Gene Deletion , Molecular Sequence Data , Mutagenesis , Phenotype , Protein Binding , Repetitive Sequences, Amino Acid , Sequence Homology, Amino AcidABSTRACT
The elongator complex is a major component of the RNA polymerase II (RNAPII) holoenzyme responsible for transcriptional elongation in yeast. Here we identify Elp3, the 60-kilodalton subunit of elongator/RNAPII holoenzyme, as a highly conserved histone acetyltransferase (HAT) capable of acetylating core histones in vitro. In vivo, ELP3 gene deletion confers typical elp phenotypes such as slow growth adaptation, slow gene activation, and temperature sensitivity. These results suggest a role for a novel, tightly RNAPII-associated HAT in transcription of DNA packaged in chromatin.
Subject(s)
Acetyltransferases/genetics , RNA Polymerase II/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Acetyltransferases/chemistry , Amino Acid Sequence , Chromatin/chemistry , Cloning, Molecular , Fungal Proteins/chemistry , Fungal Proteins/genetics , Gene Deletion , Histone Acetyltransferases , Molecular Sequence Data , Phenotype , RNA Polymerase II/chemistry , Recombinant Proteins/genetics , Saccharomyces cerevisiae/metabolism , Sequence AlignmentABSTRACT
The form of RNA polymerase II (RNAPII) engaged in transcriptional elongation was isolated. Elongating RNAPII was associated with a novel multisubunit complex, termed elongator, whose stable interaction was dependent on a hyperphosphorylated state of the RNAPII carboxy-terminal domain (CTD). A free form of elongator was also isolated, demonstrating the discrete nature of the complex, and free elongator could bind directly to RNAPII. The gene encoding the largest subunit of elongator, ELP1, was cloned. Phenotypes of yeast elp1 delta cells demonstrated an involvement of elongator in transcriptional elongation as well as activation in vivo. Our data indicate that the transition from transcriptional initiation to elongation involves an exchange of the multiprotein mediator complex for elongator in a reaction coupled to CTD hyperphosphorylation.
Subject(s)
RNA Polymerase II/genetics , Saccharomyces cerevisiae/genetics , Transcription Factors, General , Transcription Factors/genetics , Transcription, Genetic/genetics , Transcriptional Elongation Factors , Cell Division/genetics , Cloning, Molecular , Fungal Proteins/chemistry , Fungal Proteins/genetics , Gene Expression Regulation/genetics , Phenotype , Phosphorylation , Protein Binding/genetics , RNA Polymerase II/chemistry , Transcription Factors/chemistry , Transcriptional Activation/geneticsABSTRACT
Genes for the Tfb2, Tfb3, and Tfb4 subunits of yeast RNA polymerase transcription factor IIH (TFIIH) are described. All three genes are essential for cell viability, and antibodies against Tfb3 specifically inhibit transcription in vitro. A C-terminal deletion of Tfb2 caused a defect in nucleotide excision repair, as shown by UV sensitivity of the mutant strain and loss of nucleotide excision repair activity in cell extracts (restored by the addition of purified TFIIH). An interaction between Tfb3 and the Kin28 subunit of TFIIH was detected by the two-hybrid approach, consistent with a role for Tfb3 in linking kinase and core domains of the factor. The deduced amino acid sequence of Tfb2 is similar to that of the 52-kDa subunit of human TFIIH, while Tfb3 is identified as a RING finger protein homologous to the 36-kDa subunit of murine CAK (cyclin-dependent kinase activating kinase) and to the 32-kDa subunit of human TFIIH. Tfb4 is homologous to p34 of human TFIIH and is identified as the weakly associated 37-kDa subunit of the yeast factor. These and other findings reveal a one-to-one correspondence and high degree of sequence similarity between the entire set of yeast and human TFIIH polypeptides.
Subject(s)
Cyclin-Dependent Kinases/metabolism , Transcription Factors, TFII , Transcription Factors/genetics , Amino Acid Sequence , Base Sequence , Cell Survival , DNA Repair , Humans , Molecular Sequence Data , Sequence Homology, Amino Acid , Transcription Factor TFIIH , Transcription Factors/chemistryABSTRACT
Free and elongating (DNA-bound) forms of RNA polymerase II were separated from yeast. Most cellular polymerase II was found in the elongating fraction, which contained all enzyme phosphorylated on the C-terminal domain and none of the 15-subunit mediator of transcriptional regulation. These and other findings suggest that mediator enters and leaves initiation complexes during every round of transcription, in a process that may be coupled to C-terminal domain phosphorylation.
Subject(s)
RNA Polymerase II/metabolism , Transcription Factors, General , Transcription Factors, TFII , Transcription Factors/metabolism , Transcription, Genetic , Transcriptional Elongation Factors , Gene Expression Regulation, Fungal , Kinetics , Macromolecular Substances , Models, Biological , RNA Polymerase II/chemistry , RNA Polymerase II/isolation & purification , Saccharomyces cerevisiae/metabolism , Substrate Specificity , Transcription Factors/chemistry , Transcription Factors/isolation & purificationABSTRACT
Nucleotide excision repair (NER) is a biochemical process required for the repair of many different types of DNA lesions. In the yeast Saccharomyces cerevisiae, the RAD7, RAD16, and RAD23 genes have been specifically implicated in NER of certain transcriptionally repressed loci and in the nontranscribed strand of transcriptionally active genes. We have used a cell-free system to study the roles of the Rad7, Rad16, and Rad23 proteins in NER. Transcription-independent NER of a plasmid substrate was defective in rad7, rad16, and rad23 mutant extracts. Complementation studies with a previously purified NER protein complex (nucleotide excision repairosome) indicate that Rad23 is a component of the repairosome, whereas Rad7 and Rad16 proteins were not found in this complex. Complementation studies with rad4, rad7, rad16, and rad23 mutant extracts suggest physical interactions among these proteins. This conclusion was confirmed by experiments using the yeast two-hybrid assay, which demonstrated the following pairwise interactions: Rad4 with Rad23, Rad4 with Rad7, and Rad7 with Rad16. Additionally, interaction between the Rad7 and Rad16 proteins was demonstrated in vitro. Our results show that Rad7, Rad16, and Rad23 are required for transcription-independent NER in vitro. This process may involve a unique protein complex which is distinct from the repairosome and which contains at least the Rad4, Rad7, and Rad16 proteins.
Subject(s)
Adenosine Triphosphatases , DNA Repair/genetics , DNA-Binding Proteins/genetics , Fungal Proteins/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Schizosaccharomyces pombe Proteins , Transglutaminases , DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Genes, Fungal/genetics , Genetic Complementation Test , Mutation , Recombinant Fusion Proteins , Saccharomyces cerevisiae/radiation effects , Transcription, Genetic , Ultraviolet RaysABSTRACT
TFIIH is by far the most complex of the basal RNA polymerase II transcription factors. It is a protein kinase, a bi-directional DNA helicase and is essential for both transcription and nucleotide excision repair (NER). Furthermore, the factor can activate cyclin-dependent kinases and so might play a role in cell-cycle regulation. The recent elucidation of the subunit composition of TFIIH has shown an extraordinary conservation of its structure from yeast to human.
Subject(s)
DNA Repair , Transcription Factors, TFII , Transcription Factors/physiology , Transcription, Genetic , Adenosine Triphosphate/metabolism , Animals , Binding Sites , Cyclin-Dependent Kinases/metabolism , DNA Helicases/metabolism , Enzyme Activation , Humans , Phosphorylation , RNA Polymerase II/metabolism , Transcription Factor TFIIH , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
A system for rapid purification and characterization of eukaryotic topoisomerase-I mutants has been developed. The system utilizes six-histidine tagging of human topoisomerase I expressed in Saccharomyces cerevisiae to enable purification by nickel-affinity chromatography. Virtually homogenous mutant proteins are then tested for their ability to relax supercoiled DNA plasmids and their capacity for binding, cleaving and religating short defined DNA substrates. Relaxation-deficient mutants were obtained by site-directed mutagenesis of selected highly conserved amino acids. The mutants Tyr723Phe (active site mutation), Arg488Gln and Lys532Glu were inert in relaxation of DNA, whereas Lys720Glu showed a 50-fold reduction in specific relaxation activity. Accordingly, only Lys720Glu showed low, but detectable cleavage activity on suicide DNA substrates, uncoupling the cleavage and religation events of topoisomerase I. The relative religation efficiency of Lys720Glu comparable to that of wild-type topoisomerase I, indicating that Lys720 is involved in interactions important for normal DNA cleavage, but not for the religation reaction. All mutants could be cross linked by ultraviolet light to bromo-dUTP-substituted DNA oligonucleotides carrying a topoisomerase-I-binding site, indicating that the deficiency of Tyr723Phe, Arg488Gln and Lys532Glu in DNA relaxation and cleavage is not due to an inability of these mutants to bind DNA non-covalently.
Subject(s)
DNA Topoisomerases, Type I/genetics , DNA-Binding Proteins/genetics , Amino Acid Sequence , Base Sequence , DNA Primers/chemistry , DNA Topoisomerases, Type I/chemistry , DNA-Binding Proteins/chemistry , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Recombinant Proteins , Saccharomyces cerevisiae/geneticsABSTRACT
Both 45- and 47-kDa subunits of TFIIK, a subcomplex of RNA polymerase II general transcription factor TFIIH, are encoded by the yeast cyclin gene CCL1. In all likelihood, these two subunits individually form cyclin-dependent kinase/cyclin dimers with Kin28 protein, a key enzyme in phosphorylation of the C-terminal domain of RNA polymerase II concomitant with transcription.
Subject(s)
RNA Polymerase II/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , TATA-Binding Protein Associated Factors , Transcription Factor TFIID , Transcription Factors, TFII , Transcription Factors/genetics , Amino Acid Sequence , Base Sequence , Genes, Fungal , Molecular Sequence Data , Transcription Factor TFIIH , Transcription Factors/metabolismABSTRACT
The essential TFB1 and SSL1 genes of the yeast Saccharomyces cerevisiae encode two subunits of the RNA polymerase II transcription factor TFIIH (factor b). Here we show that extracts of temperature-sensitive mutants carrying mutations in both genes (tfb1-101 and ssl1-1) are defective in nucleotide excision repair (NER) and RNA polymerase II transcription but are proficient for base excision repair. RNA polymerase II-dependent transcription at the CYC1 promoter was normal at permissive temperatures but defective in extracts preincubated at a restrictive temperature. In contrast, defective NER was observed at temperatures that are permissive for growth. Additionally, both mutants manifested increased sensitivity to UV radiation at permissive temperatures. The extent of this sensitivity was not increased in a tfb1-101 strain and was only slightly increased in a ssl1-1 strain at temperatures that are semipermissive for growth. Purified factor TFIIH complemented defective NER in both tfb1-101 and ssl1-1 mutant extracts. These results define TFB1 and SSL1 as bona fide NER genes and indicate that, as is the case with the yeast Rad3 and Ss12 (Rad25) proteins, Tfb1 and Ssl1 are required for both RNA polymerase II basal transcription and NER. Our results also suggest that the repair and transcription functions of Tfb1 and Ssl1 are separable.
Subject(s)
DNA Repair/genetics , RNA Polymerase II/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , TATA-Binding Protein Associated Factors , Transcription Factor TFIID , Transcription Factors, TFII , Transcription Factors/genetics , Transcription, Genetic , Base Sequence , Dose-Response Relationship, Radiation , Fungal Proteins/genetics , Genes, Fungal/genetics , Molecular Sequence Data , Mutation , Radiation Tolerance/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/radiation effects , Subcellular Fractions/metabolism , Transcription Factor TFIIH , Ultraviolet RaysABSTRACT
Yeast TFIIH that is active in transcription can be dissociated into three components: a 5-subunit core, the SSL2 gene product, and a complex of 47 kDa, 45 kDa, and 33 kDa polypeptides that possesses protein kinase activity directed towards the C-terminal repeat domain of RNA polymerase II. These three components can reconstitute fully functional TFIIH, and all three are required for transcription in vitro. By contrast, TFIIH that is highly active in nucleotide excision repair (NER) lacks the kinase complex and instead contains the products of all other genes known to be required for NER in yeast: RAD1, RAD2, RAD4, RAD10, and RAD14. This repairosome is not active in reconstituted transcription in vitro and is significantly more active than any of the constituent polypeptides in correcting defective repair in extracts from strains mutated in NER genes.
Subject(s)
DNA Repair , Fungal Proteins/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , TATA-Binding Protein Associated Factors , Transcription Factor TFIID , Transcription Factors, TFII , Transcription Factors/metabolism , Transcription, Genetic , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Protein Kinases/metabolism , Saccharomyces cerevisiae/chemistry , Transcription Factor TFIIH , Transcription Factors/chemistry , Transcription Factors/isolation & purificationABSTRACT
Nucleotide excision repair (NER) in eukaryotes is a biochemically complex process involving multiple gene products. The budding yeast Saccharomyces cerevisiae is an informative model for this process. Multiple genes and in some cases gene products that are indispensable for NER have been isolated from this organism. Homologues of many of these yeast genes are structurally and functionally conserved in higher organisms, including humans. The yeast Rad1/Rad10 heterodimeric protein complex is an endonuclease that is believed to participate in damage-specific incision of DNA during NER. This endonuclease is also required for specialized types of recombination. The products of the RAD3, SSL2(RAD25) SSL1 and TFB1 genes have dual roles in NER and in RNA polymerase II-dependent basal transcription.
Subject(s)
DNA Repair , Mitosis/genetics , Nucleotides/genetics , RNA Polymerase II/genetics , Recombination, Genetic , Saccharomyces cerevisiae/genetics , Transcription, Genetic , Nucleotides/chemistry , Saccharomyces cerevisiae/enzymologyABSTRACT
KIN28, a member of the p34cdc2/CDC28 family of protein kinases, is identified as a subunit of yeast RNA polymerase transcription factor IIH (TFIIH) on the basis of sequence determination, immunological reactivity, and copurification. KIN28 is, moreover, one of three subunits of TFIIK, a subassembly of TFIIH with protein kinase activity directed toward the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II. Itself a phosphoprotein, KIN28 interacts specifically with the two largest subunits of RNA polymerase II. Previous work of others points to two further associations: KIN28 interacts in vivo with the cyclin CCL1, and KIN28 and CCL1 are homologous to human MO15 and cyclin H, which form the cyclin-dependent kinase-activating kinase (CAK). We show that human CAK possesses the CTD kinase activity characteristic of TFIIH.
Subject(s)
Cyclin-Dependent Kinases , Protein Serine-Threonine Kinases/genetics , RNA Polymerase II/metabolism , Saccharomyces cerevisiae Proteins , TATA-Binding Protein Associated Factors , Transcription Factor TFIID , Transcription Factors, TFII , Transcription Factors/genetics , Amino Acid Sequence , Base Sequence , Blotting, Western , Cell-Free System , Cyclins/metabolism , Humans , Molecular Sequence Data , Phosphoric Monoester Hydrolases/metabolism , Protein Binding , Protein Biosynthesis , Protein Serine-Threonine Kinases/immunology , Protein Serine-Threonine Kinases/metabolism , Recombinant Proteins/metabolism , Sequence Analysis , Sequence Homology, Amino Acid , Transcription Factor TFIIH , Transcription Factors/chemistry , Transcription Factors/metabolism , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
An RNA polymerase transcription factor IIH holoenzyme (holoTFIIH) has been resolved to near homogeneity from Saccharomyces cerevisiae. HoloTFIIH comprises the five-subunit core transcription factor described previously (Feaver, W. J., Svejstrup, J. Q., Bardwell, A. J., Bardwell, L., Buratowski, S., Gulyas, K. D., Donahue, T. F., Friedberg, E. C. and Kornberg, R. D. (1993) Cell 75, 1379-1387) and in addition, SSL2 and three further, as yet unidentified, polypeptides. HoloTFIIH possesses C-terminal repeat domain kinase activity and, together with other pure yeast transcription proteins, enables RNA polymerase II transcription in a fully defined system. By contrast, core TFIIH is inert in both C-terminal repeat domain kinase and reconstituted transcription assays.