FIGURE 2.

Bud27 is required for the optimal synthesis and maturation of 5S rRNA and tRNAs transcribed by RNA polymerase III. (A) Northern analyses of the selected pre- and mature tRNAs and 5S rRNA of wild-type and bud27Δ strains. Cells were grown in YPD medium at 30°C and shifted to 37°C for 12 h. Total RNA was extracted and equal amounts (5 µg) were subjected to northern hybridization. Probes, in brackets, are described in Supplemental Table S2. 7S pre-rRNAs are indicated by an asterisk. Note that the doublets shown in the pre-tRNA panels are different precursor forms of the corresponding tRNAs. (B) RNA Pol III occupancy (Rpb8-TAP occupancy) was analyzed by chromatin immunoprecipitation (ChIP) in the wild-type and bud27Δ cells containing a functional Rpb8-TAP tagged, which is a common subunit to the three RNA pols. Cells were grown in YPD at 30°C or shifted to 37°C for 12 h. RNA pols were precipitated using Dynabeads Pan Mouse IgG as described in Materials and Methods. Occupancy in the type 1 (5S rDNA), type 2 (tRNALeu, SUP56), and type 3 (SCR1) genes was analyzed. The values found for the immunoprecipitated PCR products were compared to those of the total input, and the ratio of each polymerase chain reaction (PCR) product of transcribed genes to a nontranscribed region of chromosome V was calculated. The average and standard deviations of three biological replicates are shown. Statistical significance, by t-Student. (**) P < 0.01, (***) P < 0.05.