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. Author manuscript; available in PMC: 2009 Dec 4.
Published in final edited form as: Methods Enzymol. 2008;449:221–237. doi: 10.1016/S0076-6879(08)02411-7

Figure 1. tRNAVal(AAC) levels and aminoacylation levels both decrease at 37 °C in a trm8-Δ trm4-Δ strain.

Figure 1

(A) Northern analysis of tRNA levels and aminoacylation. Strains were grown in YPD at 28 °C to OD600 ~1.3, rapidly shifted to 37 °C, and cells were harvested by filtration at the indicated times. 2 µg of RNA isolated under acidic conditions was separated on an acidic gel and analyzed by Northern blotting. For each strain, one sample was deacylated prior to gel electrophoresis. Dashed and solid arrows indicate aminoacylated and deacylated tRNA species, respectively. Note that tRNAVal(AAC) from strains lacking TRM8 migrates faster than wild type. (B) Quantification of the levels of tRNAVal(AAC). The ordinate shows the ratio of the levels of tRNAVal(AAC) at each time point relative to its level in the wild type strain immediately before temperature shift (each value itself first normalized to 5S RNA). (C) Quantification of the aminoacylation levels of tRNAVal(AAC).