ABSTRACT
The transcription factor TFIID is a large multiprotein complex, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), which plays a key role in the regulation of gene expression by RNA polymerase II. The three-dimensional structure of yeast (y) TFIID, determined at approximately 3 nm resolution by electron microscopy and image analysis, resembles a molecular clamp formed by three major lobes connected by thin linking domains. The yTFIID is structurally similar to the human factor although the clamp appears more closed in the yeast complex, probably reflecting the conformational flexibility of the structure. Immunolabelling experiments showed that nine TAFs that contain the histone fold structural motif were located in three distinct substructures of TFIID. The distribution of these TAFs showed that the previously reported pair-wise interactions between histone fold domain (HFD)-containing TAFs are likely to occur in the native yTFIID complex. Most of the HFD-containing TAFs have been found in two distinct lobes, thus revealing an unexpected and novel molecular organization of TFIID.
Subject(s)
Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Transcription Factors, TFII/chemistry , Transcription Factors/chemistry , Amino Acid Motifs , Animals , Dimerization , Drosophila Proteins/chemistry , Histones/chemistry , Humans , Image Processing, Computer-Assisted , Macromolecular Substances , Microscopy, Electron , Microscopy, Immunoelectron , Models, Molecular , Multiprotein Complexes , Precipitin Tests , Protein Conformation , Protein Interaction Mapping , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/ultrastructure , Species Specificity , Structure-Activity Relationship , Transcription Factor TFIID , Transcription Factors/classification , Transcription Factors/ultrastructure , Transcription Factors, TFII/ultrastructureABSTRACT
The RNA polymerase II transcription factor TFIID, composed of the TATA-binding protein (TBP) and TBP-associated factors (TAF(II)s), nucleates preinitiation complex formation at protein-coding gene promoters. SAGA, a second TAF(II)-containing multiprotein complex, is involved in transcription regulation in Saccharomyces cerevisiae. One of the essential protein components common to SAGA and TFIID is yTAF(II)25. We define a minimal evolutionarily conserved 91-amino-acid region of TAF(II)25 containing a histone fold domain that is necessary and sufficient for growth in vivo. Different temperature-sensitive mutations of yTAF(II)25 or chimeras with the human homologue TAF(II)30 arrested cell growth at either the G(1) or G(2)/M cell cycle phase and displayed distinct phenotypic changes and gene expression patterns. Immunoprecipitation studies revealed that TAF(II)25 mutation-dependent gene expression and phenotypic changes correlated at least partially with the integrity of SAGA and TFIID. Genome-wide expression analysis revealed that the five TAF(II)25 temperature-sensitive mutant alleles individually affect the expression of between 18 and 33% of genes, whereas taken together they affect 64% of all class II genes. Thus, different yTAF(II)25 mutations induce distinct phenotypes and affect the regulation of different subsets of genes, demonstrating that no individual TAF(II) mutant allele reflects the full range of its normal functions.
Subject(s)
DNA-Binding Proteins/genetics , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Mutation/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , TATA-Binding Protein Associated Factors , Transcription Factors, TFII/chemistry , Transcription Factors, TFII/genetics , Base Composition , Blotting, Western , Cell Cycle , Chromosome Mapping , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Flow Cytometry , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Humans , Macromolecular Substances , Multiprotein Complexes , Oligonucleotide Array Sequence Analysis , Phenotype , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Temperature , Transcription Factor TFIID , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription Factors, TFII/metabolism , Two-Hybrid System TechniquesABSTRACT
The efficient assembly of newly replicated and repaired DNA into chromatin is essential for proper genome function. Based on genetic studies in Saccharomyces cerevisiae, the histone chaperone anti-silencing function 1 (Asf1) has been implicated in the DNA repair response. Here, the human homologs are shown to function synergistically with human CAF-1 to assemble nucleosomes during nucleotide excision repair in vitro. Furthermore, we demonstrate that hAsf1 proteins can interact directly with the p60 subunit of hCAF-1. In contrast to hCAF-1 p60, the nuclear hAsf1 proteins are not significantly associated with chromatin in cells before or after the induction of DNA damage, nor specifically recruited to damaged DNA during repair in a bead-linked DNA assay. A model is proposed in which the synergism between hAsf1 and CAF-1 for nucleosome formation during DNA repair is achieved through a transient physical interaction allowing histone delivery from Asf1 to CAF-1.
