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. 2012 Jul 2;287(34):29021–29034. doi: 10.1074/jbc.M112.380147

FIGURE 5.

FIGURE 5.

Stability of selected glucose-repressible mRNAs in derepressing growth conditions after inhibiting Snf1as, addition of glucose, or inactivating RNA pol II (rpb1-1) at 36. 5 °C. Strain TYY1077 (SNF1as) was grown in YPD medium and derepressed in YP containing 0.05% glucose as described under “Experimental Procedures.” Either glucose (D, final concentration 3%) and 2NM-PP1 (I, final concentration 10 μm) was added 4 h after initial glucose depletion, and samples were removed 5 and 15 min after glucose addition or 7.5 and 22 min after 2NM-PP1 addition. For inactivation of RNA pol II, strain KBY108 (rpb1-1 SNF1as) was grown a 25 °C, and the temperature was rapidly raised to 36.5 °C as described under “Experimental Procedures.” Aliquots were removed for mRNA analysis 20, 40, and 60 min later. RT-qPCR was performed as described under “Experimental Procedures.” The RNA was converted to cDNA using random hexamers and quantified by qPCR. The mRNA values were normalized to the amount of 18 S ribosomal RNA (measured as cDNA) and are expressed relative to the amount of mRNA present in the absence of 2NM-PP1 or glucose for TYY1077 or relative to the amount of mRNA present immediately before heat inactivation of RNA pol II for strain KBY108 (rpb1-1 ts).