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. 2017 Jan 24;45(8):4431–4451. doi: 10.1093/nar/gkx037

Figure 2.

Figure 2.

Pol II catalytic mutants generally decrease in vivo gene expression. (A) Pol II mutant effects on gene expression are exacerbated with increasing promoter strength. Steady-state RNA levels of reporter genes used for Pol II occupancy experiments (GAL1p::YLR454w and TEF1p::YLR454w) and endogenous TEF1 levels from cells grown in galactose- or glucose-containing medium as indicated. Values were normalized to WT TEF1p::YLR454w expression level in YPD. The most severe expression defects are evident at GAL1p::YLR454w for both GOF and LOF mutants compared to WT. Individual data points from at least three biological repeats are shown with error bars indicating average +/− standard deviation of the mean. (B) Endogenous GAL1 mRNA expression level is decreased in Pol II catalytic mutant strains compared to WT, yet GAL1 expression defects in mutants are less severe than GAL1p::YLR454w expression defects as showed in A. Values were normalized to WT GAL1 mRNA level. Error bars as in A. (C) Pol II catalytic mutants delay induction of the GAL1 promoter. Time courses showing induction of GAL1p::YLR454w and endogenous GAL1 mRNA in WT and Pol II catalytic mutants. Overnight grown cells were inoculated into fresh YPRaf medium and grown until mid-log phase at 30°C, subsequently galactose was added (4% final concentration) to induce GAL gene expression. RNAs isolated prior (time 0) and after galactose addition were used for northern blotting to determine accumulation of mature GAL1p::YLR454w and endogenous GAL1. Data normalized to WT 120 min value and plotted using non-linear regression using GraphPad Prism. Individual data points from at least three biological repeats are shown.