Fig. 3.
Tb-TrpRS1 and Tb-TrpRS2 are essential for the growth of procyclic T. brucei and are responsible for formation of tryptophanyl-tRNA in the cytosol and the mitochondria, respectively (A) Growth curve in the presence and absence of tetracycline (+Tet and −Tet) of a representative clonal T. brucei RNAi cell line ablated for Tb-TrpRS1. (A Inset) A Northern blot for Tb-TrpRS1 mRNA. The time of sampling is indicated by the arrow. The rRNAs in the lower panel serve as loading controls. (B) Same as A for an RNAi cell line ablated for Tb-TrpRS2. (C) Northern blot analysis of total and mitochondrial RNA isolated under acidic conditions from the Tb-TrpRS1 RNAi cell line. The total RNA fraction only contains ≈5% of mitochondrial RNA and, thus, essentially represents cytosolic RNA. Days of induction (0 and 3) by tetracycline are indicated at the bottom. The blots were probed for the T. brucei tRNATrp as well as tRNALeu and tRNATyr, which serve as controls not affected by the RNAi. The RNA fractions were resolved on long acid urea gels, which allow separation of aminoacylated from deacylated tRNAs. The bar graph shows the quantification of the results. Relative amounts of aminoacylated tRNAs are indicated for the tRNATrp and the controls (tRNALeu and tRNATyr), respectively. For each lane, the sum of aminoacylated and deacylated tRNA was set to 100%. (D) Same as C, but analysis was done for the Tb-TrpRS2 RNAi cell line.