Structure/Function Analysis of the Phosphatidylinositol-3-Kinase Domain of Yeast Tra1

Abstract

Tra1 is an essential component of the Saccharomyces cerevisiae SAGA and NuA4 complexes. Using targeted mutagenesis, we identified residues within its C-terminal phosphatidylinositol-3-kinase (PI3K) domain that are required for function. The phenotypes of tra1-P 3408 A, S 3463 A, and SRR 3413-3415 AAA included temperature sensitivity and reduced growth in media containing 6% ethanol or calcofluor white or depleted of phosphate. These alleles resulted in a twofold or greater change in expression of $7% of yeast genes in rich media and reduced activation of PHO5 and ADH2 promoters. Tra1-SRR 3413 associated with components of both the NuA4 and SAGA complexes and with the Gal4 transcriptional activation domain similar to wild-type protein.Tra1-SRR 3413 was recruited to the PHO5 promoter in vivo but gave rise to decreased relative amounts of acetylated histone H3 and histone H4 at SAGA and NuA4 regulated promoters. Distinct from other components of these complexes, tra1-SRR 3413 resulted in generation-dependent telomere shortening and synthetic slow growth in combination with deletions of a number of genes with roles in membrane-related processes. While the tra1 alleles have some phenotypic similarities with deletions of SAGA and NuA4 components, their distinct nature may arise from the simultaneous alteration of SAGA and NuA4 functions.

Analysis of Gal4-directed transcription activation using Tra1 mutants selectively defective for interaction with Gal4

“…Toward this end, we first identified Tra1-dependent genes by comparing the mRNA population of a WT TRA1 strain to that of a strain bearing a temperaturesensitive tra1 allele (tra1-2 ts ) (31) under nonpermissive conditions. After inactivation of Tra1, ≈3% of yeast genes were down-regulated greater than twofold (Dataset S1), consistent with a previous study that analyzed other tra1 alleles (32).…”

Domains of Tra1 Important for Activator Recruitment and Transcription Coactivator Functions of SAGA and NuA4 Complexes

“…This mutation shows that Tra1 somehow influences either specificity or catalytic activity of the HAT subunit without disrupting the association of HAT module components Ada2 and Ada3. It was previously found that several mutations within the PI3K-like domain of Tra1 showed in vivo defects in histone acetylation but no defect in Tra1 recruitment (61). Our initial experiments show that SAGA containing TRA1 ⌬32 has normal activity in acetylating recombinant H3 at residue K9.…”

“…Tra1 and its human homolog TRRAP are catalytically inactive since they lack several critical residues necessary for protein phosphorylation, but they retain the structural fold of the PI3K domain (61,76). TRRAP is a functional target of oncogenic transcription factors that include myc, E2F, and E1a (13,22,46,48).…”

Transcriptional Regulation inSaccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

“…Tra1 and its human homolog, TRRAP, are members of the PI3-related protein kinase family, but Tra1 and TRRAP have specifically lost kinase activity (Mutiu et al 2007). Biochemical and genetic experiments showed that several activators, including Gcn4 and Gal4, interact with Tra1 and that this interaction is important for activated transcription of SAGAdependent genes (Brown et al 2001;Fishburn et al 2005;Reeves and Hahn 2005).…”