RESUMO
We have purified and characterized a Gcn5-independent nucleosomal histone H3 HAT complex, NuA3 (Nucleosomal Acetyltransferase of histone H3). Peptide sequencing of proteins from the purified NuA3 complex identified Sas3 as the catalytic HAT subunit of the complex. Sas3 is the yeast homolog of the human MOZ oncogene. Sas3 is required for both the HAT activity and the integrity of the NuA3 complex. In addition, NuA3 contains the TBP- associated factor, yTAF(II)30, which is also a component of the TFIID, TFIIF, and SWI/SNF complexes. Sas3 mediates interaction of the NuA3 complex with Spt16 both in vivo and in vitro. Spt16 functions as a component of the yeast CP (Cdc68/Pob3) and mammalian FACT (facilitates chromatin transcription) complexes, which are involved in transcription elongation and DNA replication. This interaction suggests that the NuA3 complex might function in concert with FACT-CP to stimulate transcription or replication elongation through nucleosomes by providing a coupled acetyltransferase activity.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Transporte/metabolismo , Domínio Catalítico , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID , Fatores de Transcrição/metabolismo , Acetiltransferases/química , Acetiltransferases/genética , Acetiltransferases/isolamento & purificação , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica , Células HeLa , Histona Acetiltransferases , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Peso Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Mutação/genética , Testes de Precipitina , Ligação Proteica , Proteínas Quinases/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Transcrição Gênica/genética , Fatores de Elongação da Transcrição , Técnicas do Sistema de Duplo-HíbridoRESUMO
Homologs of the chromatin-bound yeast silent information regulator 2 (SIR2) protein are found in organisms from all biological kingdoms. SIR2 itself was originally discovered to influence mating-type control in haploid cells by locus-specific transcriptional silencing. Since then, SIR2 and its homologs have been suggested to play additional roles in suppression of recombination, chromosomal stability, metabolic regulation, meiosis, and aging. Considering the far-ranging nature of these functions, a major experimental goal has been to understand the molecular mechanism(s) by which this family of proteins acts. We report here that members of the SIR2 family catalyze an NAD-nicotinamide exchange reaction that requires the presence of acetylated lysines such as those found in the N termini of histones. Significantly, these enzymes also catalyze histone deacetylation in a reaction that absolutely requires NAD, thereby distinguishing them from previously characterized deacetylases. The enzymes are active on histone substrates that have been acetylated by both chromatin assembly-linked and transcription-related acetyltransferases. Contrary to a recent report, we find no evidence that these proteins ADP-ribosylate histones. Discovery of an intrinsic deacetylation activity for the conserved SIR2 family provides a mechanism for modifying histones and other proteins to regulate transcription and diverse biological processes.
Assuntos
Proteínas Fúngicas/fisiologia , Inativação Gênica/fisiologia , Histona Desacetilases/fisiologia , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/enzimologia , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Transativadores/fisiologia , Acetilação , Adenosina Difosfato Ribose/metabolismo , Animais , Galinhas , Proteínas Fúngicas/genética , Histona Desacetilases/genética , Histonas/química , Lisina/metabolismo , Família Multigênica , NAD/metabolismo , Niacinamida/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Sirtuína 2 , Sirtuínas , Transativadores/genéticaRESUMO
We report the crystal structure of the yeast protein Hpa2 in complex with acetyl coenzyme A (AcCoA) at 2.4 A resolution and without cofactor at 2.9 A resolution. Hpa2 is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, a family of enzymes with diverse substrates including histones, other proteins, arylalkylamines and aminoglycosides. In vitro, Hpa2 is able to acetylate specific lysine residues of histones H3 and H4 with a preference for Lys14 of histone H3. Hpa2 forms a stable dimer in solution and forms a tetramer upon binding AcCoA. The crystal structure reveals that the Hpa2 tetramer is stabilized by base-pair interactions between the adenine moieties of the bound AcCoA molecules. These base-pairs represent a novel method of stabilizing an oligomeric protein structure. Comparison of the structure of Hpa2 with those of other GNAT superfamily members illustrates a remarkably conserved fold of the catalytic domain of the GNAT family even though members of this family share low levels of sequence homology. This comparison has allowed us to better define the borders of the four sequence motifs that characterize the GNAT family, including a motif that is not discernable in histone acetyltransferases by sequence comparison alone. We discuss implications of the Hpa2 structure for the catalytic mechanism of the GNAT enzymes and the opportunity for multiple histone tail modification created by the tetrameric Hpa2 structure.
Assuntos
Acetiltransferases/química , Proteínas de Ligação a DNA , Proteínas Fúngicas/química , Família Multigênica , Proteínas Quinases/química , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Acetilação , Acetiltransferases/metabolismo , Adenina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Pareamento de Bases , Sítios de Ligação , Domínio Catalítico , Sequência Conservada , Cristalização , Cristalografia por Raios X , Dimerização , Proteínas Fúngicas/metabolismo , Histona Acetiltransferases , Histonas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Proteínas Quinases/metabolismo , Estrutura Quaternária de Proteína , Relação Estrutura-AtividadeRESUMO
We have solved the crystal structure of the yeast histone acetyltransferase Hat1-acetyl coenzyme A (AcCoA) complex at 2.3 A resolution. Hat1 has an elongated, curved structure, and the AcCoA molecule is bound in a cleft on the concave surface of the protein, marking the active site of the enzyme. A channel of variable width and depth that runs across the protein is probably the binding site for the histone substrate. A model for histone H4 binding by Hat1 is discussed in terms of possible sources of specific lysine recognition by the enzyme. The structure of Hat1 provides a model for the structures of the catalytic domains of a protein superfamily that includes other histone acetyltransferases such as Gcn5 and CBP.
