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

Figure 8.

Figure 8.

Pol II catalytic mutants defective for IMD2 transcription do not abolish GTP sensing. (A) (Top left) Schematic of IMD2 gene transcription in presence and absence of MPA. In GTP-replete cells, IMD2 is transcribed from upstream ‘G’ start sites producing non-functional CUTs, which are degraded by the RNA exosome. In the presence of MPA (GTP depletion), a downstream ‘A’ start site is utilized to produce functional IMD2 transcripts. (top right) MPA depletes GTP by inhibition of IMPDH activity, present in yeast in two paralogous enzymes, Imd3 and Imd4. Upon GTP depletion, expression of an MPA-resistant IMPDH enzyme, Imd2 is induced. (bottom) Schematic of the imd2Δ::HIS3 construct and expected phenotype upon MPA treatment in synthetic medium lacking leucine or histidine. All strains lack endogenous IMD2 ORF, which is replaced with HIS3 (imd2Δ::HIS3), hence rendering them highly sensitive to MPA treatment. (B) In absence of endogenous IMD2, Pol II catalytic mutants respond to GTP depletion similarly to WT. 10-fold serial dilutions of saturated cultures of Pol II catalytic mutants plated on synthetic medium lacking leucine for comparison of growth in various concentration of MPA treatment at 30°C. (Upper panel) For the Pol II catalytic mutants’ response to MPA treatment, the most obvious comparison with WT can be made at 1 μg/ml MPA treatment (highlighted with red box). (Lower panel) Addition of guanine suppresses MPA sensitivity of Pol II mutants (highlighted with red boxes). (C) 10-fold serial dilutions of saturated cultures of Pol II catalytic mutants plated on synthetic medium lacking histidine for comparison of growth at various concentrations of MPA treatment at 30°C. 3-aminotriazole (3-AT) is a competitive inhibitor of His3p and can be used to assess level of HIS3 expression. Slow catalytic mutants constitutively use the downstream, productive ‘A’ site at the IMD2 promoter, which produces a functional transcript (see text for details). Hence, His+ colonies are observed even in the absence of MPA (red highlighted box). All Pol II catalytic mutants show inducible His+ phenotype upon low level of MPA treatment (highlighted black box), indicating retention of GTP sensing ability.