Table 2

Inhibition of GCN4 expression in yeast cells overexpressing pk2 or a truncated GCN2 kinase domain

Overexpressed proteinGCN4-lacZ expression, units
GCN2gcn2Δ
RDRRDR
None (vector)1011066
PK2103467
GCN2-TK94066
PK2-tag934NDND
  • Plasmids expressing pk2, gcn2-tk, or a c-myc epitope tagged version of pk2 were introduced into isogenic Mata ura3-52 leu2-3,-112 trpl-Δ63 GCN4-lacZ, TRP1〉 strains containing wild-type GCN2 (H1642) or lacking GCN2 (gcn2Δ, H1895). For repressing (R) conditions, transformants were grown for approximately 9.5 h in synthetic minimal medium containing 10% galactose and 2% raffinose. For derepressing (DR) conditions, cultures were first grown for 2 h under repressing conditions and then 3-aminotriazole, an inhibitor of histidine biosynthesis, was added to 10 mM and the cultures were incubated for another 7.5 h. Cell harvesting and β-galactosidase assays were performed as described (41, 42), and β-galactosidase activities are expressed as nanomoles of o-nitrophenyl β-d-galactopyranoside hydrolyzed per min per mg of protein. Results are the average of two or three transformants, and the individual measurements deviated from the average values shown here by 29% or less. ND, not determined.