Title

Identification and molecular characterization of a type-A histone acetyltransferase from Tetrahymena thermophila

Date of Award

1996

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Advisor(s)

C. David Allis

Keywords

chromatin, posttranslational acetylation, Cellular biology, Molecular biology

Subject Categories

Cell Biology

Abstract

Gene expression in eukaryotes requires the coordination of at least three events; the binding of activators and co-activators to DNA enhancer elements, the recruitment of the basal transcription apparatus to DNA promoter elements, and the relief of repressive chromatin structure. Indirect evidence has long suggested that posttranslational acetylation of the four core histones, the primary protein components of chromatin, alters chromatin structure and potentiates transcription. In this dissertation, I describe the isolation and cloning of a catalytically active subunit (p55) of type-A histone acetyltransferase (HAT) from Tetrahymena, an enzyme that catalyzes transcription-related acetylation.

The sequence of Tetrahymena p55 reveals striking homology to the highly conserved yeast co-activator GCNS, which, like p55, possesses intrinsic histone acetyltransferase activity. Amino acid sequence analyses of Tetrahymena p55 and its counterparts in yeast, Drosophila, and humans reveals several conserved domains, including single copies of the bromodomain. The bromodomain is a highly conserved motif found in an extremely limited number of polypeptides with related functions in activated gene expression. Thus the bromodomain may provide a mechanism for targeting histone acetyltransferase and related activities to individual genes within specific chromatin domains.

Analyses of Tetrahymena macronuclear extracts suggest that p55 is a component of a multimeric enzyme complex with a molecular mass of approximately 220 kDa. This value agrees closely with the theoretical mass of a predicted complex in yeast containing the gene products of GCN5, ADA1, ADA2, ADA3, and ADA5, suggesting that both the Tetrahymena and yeast HAT A activities are composed of similar heteromeric complexes. Together with independent genetic and biochemical evidence implicating Gcn5p, Ada1p, Ada2p, Ada3p, and Ada5p as components of a putative transcriptional adaptor complex necessary for the full activity of several genes in yeast, these findings establish a direct link between histone acetyltransferase activity and factors required for activated gene expression. Moreover, defining a biochemical function for yGcn5p and its homologs as HATs implies that other transcriptional co-activators may similarly affect gene expression by directly engaging and modifying chromatin components.

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