Figure 7.
The catalytically inactive mutant Rat1D235A does not correct defects in 5.8S rRNA maturation and Pol I termination when Rat1 is depleted. (A) PMET3-RAT1 strains transformed with plasmid without insert (vector), with RAT1 (wild type [WT]), or with rat1 (D235A) were tested for growth at 30°C on SD-methionine or SD + 5 mM methionine. (B) Western blot for detection of tagged Rat1 or Rat1D235A with HA3. Nop1 detection was used as a loading control. (C) Northern analysis of 5.8S rRNA maturation. RNAs were extracted as described in Figure 1B. Total RNA was resolved on an 8% polyacrylamide/urea gel. Mature 5.8SS and 5.8SL rRNAs were detected with probe 017 (see Fig. 1A; Supplemental Table S2). (D–F) TRO analysis over the rDNA repeats in strains PMET3-RAT1 + vector (D), PMET3-RAT1 + p-RAT1 (E), and PMET3-RAT1 + p-rat1 (D235A) (F). Primers and quantifications are as in Figure 5. The mean of three independent experiments is shown with standard deviation. (G–I) Profiles of Pol I cross-linking over the intergenic region of rDNA repeats in strains PMET3-RAT1 + vector (G), PMET3-RAT1 + p-RAT1 (H), and PMET3-RAT1 + p-rat1 (D235A) (I). Primers and quantifications are as described in Figure 6. A representative graph of two independent experiments is shown.