Strains used were derived from W303-1a, with PMET3::RAT1 integrated at the RAT1 locus and carrying a URA3 plasmid (pRS316) to allow growth in medium lacking uracil. Plasmids used are listed in Table 1 . The effects of Rat1 depletion were analyzed in this strain additionally transformed with pRS315 (empty plasmid; strain YEAH212), pRS315-RAT1-HA (expressing functional HA-tagged Rat1; strain YEAH213) or pRS315-rat1(D235A)-HA (expressing catalytically inactive, HA tagged Rat1D235A; strain YEAH214).
Following addition of methionine for 8 h, the Rat1-depleted and complemented strains were pulse-labeled with [3H-5,6] uracil. Cells were harvested at 30 sec intervals by transfer of 900 µl culture samples into 900 µl ethanol at −80°C, to rapidly inhibit label uptake and RNA metabolism. RNA was extracted, separated on denaturing agarose/glyoxal gels and transferred to nylon membranes (Hybond N+). RNA labeled with [3H] uracil was visualized using a Fuji imager (Figure S1 ). To allow different data sets to be directly compared, signals were normalized to the average values for the 27SA pre-rRNA plateau, which was previously shown to give the most reliable results [7] (link).
Following addition of methionine for 8 h, the Rat1-depleted and complemented strains were pulse-labeled with [3H-5,6] uracil. Cells were harvested at 30 sec intervals by transfer of 900 µl culture samples into 900 µl ethanol at −80°C, to rapidly inhibit label uptake and RNA metabolism. RNA was extracted, separated on denaturing agarose/glyoxal gels and transferred to nylon membranes (Hybond N+). RNA labeled with [3H] uracil was visualized using a Fuji imager (
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