Project description:Tra1 is a component of the Saccharomyces cerevisiae SAGA and NuA4 complexes and a member of the phosphatidylinositol 3-kinase (PI3K) related kinase family that contain a C- terminal PI3K domain followed by a ~ 35-residue FATC domain. We have characterized four alleles with single residue changes in the FATC domain. Of these tra1-L3733A had the most pronounced effects with phenotypes including temperature and cold sensitivity, and reduced growth in media containing ethanol, Calcofluor white, rapamycin, chloramphenicol and geneticin. Tra1-L3733A interacted at normal levels with components of the NuA4 and SAGA complexes, and did not significantly alter histone acetylation patterns. The tra1-L3733A allele resulted in two-fold or greater change in expression of approximately 11% of yeast genes in rich media. Of the 279 genes with increased expression, 175 were ribosomal subunits or involved in ribosomal function or biogenesis. Elevated levels of Pol I and Pol III transcripts were also observed. The phenotypes of the tra1-L3733A overlapped with but were not identical to strains containing deletions of SAGA or NuA4 components or with strains containing mutations in the PI3K domain. Our finding that the double mutant allele, tra1-SRR3413/L3733A with alterations in the PI3K and FATC domains, resulted in wild type growth, suggests a model whereby the FATC domain negatively regulates the activity of the PI3K domain. Expression of genes involved in ribosome biosynthesis, other than the ribosomal subunits themselves, returned to near normal levels in the double mutant strain. We also characterized tra1-G3745, which contains an additional glycine residue following the normal C-terminal phenylalanine. This allele did not support viability and showed severe dominant negative effects. In contrast to what was observed for tra1-L3733A, tra1-G4745 resulted in decreased expression of genes required for ribosome biogenesis and did not interact with Esa1 or Spt7.
Project description:Tra1 is a component of the Saccharomyces cerevisiae SAGA and NuA4 complexes and a member of the phosphatidylinositol 3-kinase (PI3K) related kinase family that contain a C- terminal PI3K domain followed by a ~ 35-residue FATC domain. We have characterized four alleles with single residue changes in the FATC domain. Of these tra1-L3733A had the most pronounced effects with phenotypes including temperature and cold sensitivity, and reduced growth in media containing ethanol, Calcofluor white, rapamycin, chloramphenicol and geneticin. Tra1-L3733A interacted at normal levels with components of the NuA4 and SAGA complexes, and did not significantly alter histone acetylation patterns. The tra1-L3733A allele resulted in two-fold or greater change in expression of approximately 11% of yeast genes in rich media. Of the 279 genes with increased expression, 175 were ribosomal subunits or involved in ribosomal function or biogenesis. Elevated levels of Pol I and Pol III transcripts were also observed. The phenotypes of the tra1-L3733A overlapped with but were not identical to strains containing deletions of SAGA or NuA4 components or with strains containing mutations in the PI3K domain. Our finding that the double mutant allele, tra1-SRR3413/L3733A with alterations in the PI3K and FATC domains, resulted in wild type growth, suggests a model whereby the FATC domain negatively regulates the activity of the PI3K domain. Expression of genes involved in ribosome biosynthesis, other than the ribosomal subunits themselves, returned to near normal levels in the double mutant strain. We also characterized tra1-G3745, which contains an additional glycine residue following the normal C-terminal phenylalanine. This allele did not support viability and showed severe dominant negative effects. In contrast to what was observed for tra1-L3733A, tra1-G4745 resulted in decreased expression of genes required for ribosome biogenesis and did not interact with Esa1 or Spt7. Three biological replicate experiments including one dye-swap were performed for yeast strains CY3003(TRA1::Tn10LUK with IB150(myc9-tra1_L3733A-YCplac111)) and CY3015(TRA1::Tn10LUK with IB157(myc9-tra1_SRR3413_L3733A-YCplac111)) with reference to CY2706(TRA1::Tn10LUK with 1980(myc9-TRA1-YCplac111)). Similarly, three biological replicate experiments including one dye-swap were performed for yeast strain CY3019(TRA1::Tn10LUK with 1259(myc-TRA1-YCplac111) and IB162(myc9-tra1_G3745-YCplac111)) with reference to CY3020(TRA1::Tn10LUK with 1259(myc-TRA1-YCplac111) and IB160(myc9-TRA1-YCplac111)).
Project description:Tra1 is an essential component of both the yeast SAGA/SLIK and NuA4 complexes, where it recruits these complexes to acetylate histones at targeted promoters. Importantly, Tra1 regulates the transcriptional response to multiple stresses. This submission contains RNA sequencing data from Saccharomyces cerevisiae strains expressing a Tra1 mutant with three arginine to glutamine mutations in the PI3K domain (tra1Q3; Berg et al., 2018). Our goal was to identify how gene expression profile changes during the chronological aging process due to mutation in the PI3K domain of Tra1.
Project description:Tra1 is an essential component of the SAGA and NuA4 transcriptional co-activator complexes and is linked to multiple cellular processes associated with the yeast response to antifungal drugs. This submission contains RNA sequencing data from Saccharomyces cerevisiae strains expressing a Tra1 mutant with three arginine to glutamine mutations in the PI3K domain (tra1Q3; Berg et al., 2018). Our goal was to identify how gene expression changes due to mutation to the PI3K domain of Tra1.
Project description:Tra1 is an essential component of the S. 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 phenotype of tra1-P3408A, S3463A and SRR3413-3415AAA included temperature sensitivity and reduced growth in media containing 6% ethanol or aminotriazole. These alleles resulted in ≥2-fold change in expression of ~7% of yeast genes in rich media and reduced activation of PHO5 and ADH2 promoters. Tra1-SRR3413 associated with components of both the NuA4 and SAGA complexes and with the Gal4 transcriptional activation domain similar to wild-type protein. Tra1-SRR3413 resulted in a decreased ratio of acetylated histone H3 and H4 to total histone H3 at the PHO5 promoter that appeared largely due to increased unacetylated histone. Slow growth of a tra1-SRR3413 strain in aminotriazole was suppressed by deletion of Vps1 and the Vps1 interacting protein, Rad16. In addition, ethanol sensitivity was suppressed by loss of Vam3, suggesting that Tra1 is involved with protein trafficking and membrane sorting. Furthermore, tra1-SRR3413 results in generation dependent telomere shortening. While the tra1 alleles have some phenotypic similarities with deletions of SAGA and/or NuA4 components, the pattern of gene expression, genetic interactions and telomere shortening suggest possible novel functions for the PI3K-domain of Tra1. Keywords: yeast, Tra1, PI3-kinase domain, gene expression, genetic modification
Project description:Tra1 is a component of the Saccharomyces cerevisiae SAGA and NuA4 complexes and a member of the phosphatidylinositol 3-kinase kinase family that contain a C-terminal PI3K domain followed by a 35-residue FATC domain. We analyzed alleles with single residue changes in the FATC domain. Of these tra1-L3733A had the most pronounced effects, resulting in phenotypes similar but not identical to those resulting from mutations in the PI3K domain or SAGA or NuA4 components. To analyze the transcriptional effects of the tra1-L3733A allele, we performed gene profiling of wild-type and tra1-L3733A strains. Expression of 11 genes was elevated ≥ 2-fold, 79 genes were decreased ≥ 2-fold. Of the 11 genes with elevated expression, four (HSP26, PIR3, DDR2 and GRE1) have roles in cellular stress response. The only gene ontology process significantly enriched in the genes with decreased expression greater than 2-fold was lysine biosynthesis (GO: 0009085). Broader analysis of gene ontology processes revealed expression of genes involved in cellular respiration (GO:0045333) and the overlapping process generation of precursor metabolites and energy (GO: 0006091) showed an average upward shift of ~10% relative to the total collection of genes. A similar trend of less magnitude was seen for vitamin metabolic processes (GO:0006766). A slight downward shift was seen for genes involved in translation (GO:0006412) and membrane organization (GO:0016044). Examination of gene expression changes in tra1-L3733A
Project description:The emergence of drug-resistant fungal strains is a threat to human health. Identifying targets that increase the susceptibility of fungal pathogens to current therapies will significantly improve outcomes. Tra1 is an essential component of the SAGA and NuA4 transcriptional co-activator complexes and is linked to multiple cellular processes associated with the yeast response to antifungal drugs. This submission contains RNA sequencing data from Candida albicans strain expressing a Tra1 mutant with three arginine to glutamine mutations in the PI3K domain (tra1Q3; Berg et al., 2018) treated with or without the antifungal caspofungin. Our goal was to identify genes regulated by Tra1 in C. albicans and identify genes with differential responses in the mutant tra1 strain upon capsofungin relative to the wild-type strain. We identified 68 genes that were differentially expressed when the tra1Q3 strain was treated with caspofungin, as compared to gene expression changes induced by either tra1Q3 or caspofungin alone. Included in this set were genes involved in cell wall maintenance, adhesion and filamentous growth. As a critical component of both SAGA and NuA4, Tra1 is uniquely positioned to regulate the yeast antifungal response and emerges, through targeting its PI3K domain, as a promising new therapeutic target for fungal infections.