tRNATrp precursors accumulate in trm10Δ strains and in TRM10 strains grown in 5FU. (A) Diagram indicating the three pre-tRNATrp species detected by northern probes. Orange and yellow lines indicate the hybridization of 5′ leader probes (GAT and GTT, indicating the last 3 nt of the targeted leader sequence, respectively), while green lines indicate the hybridization of the intron probe (sequences listed in Supplemental Table S2). Images are not to scale, but bent lines indicate probe sequences that lose hybridization to intron-containing and mature tRNAs. (B) Northern analysis of RNA derived from the indicated strains using 5'-biotinylated probes and visualized with chemiluminescence. The bar to the left of each set of results indicates the probe that was utilized (colored as in A). (C) Quantification of relative total pre-RNA levels (for all pre-tRNA species combined) from strains shown in B. Relative tRNA levels were calculated by comparing the observed intensity for each RNA to the normalized abundance observed in the trm10Δ strain plus tRNATrp overexpression (lane 5), to allow for quantifying the full range of increased and decreased pre-tRNA levels. Duplicate data were plotted, with each end of the error bars showing each data point.
