FIGURE 5.

Parameters of E. coli cells harboring wt-trmD and S88L-trmD alleles. A–D, colony forming unit (CFU) analysis of growth (in red) and intracellular activity analysis of TrmD (in blue) of the wt-trmD allele at 30 °C (A), the S88L-trmD allele at 30 °C (B), the wt-trmD allele at 43 °C (C), and the S88L-trmD allele at 43 °C (D). The data are shown as average of two independent experiments. The error bars represent standard deviations. E, Western analysis of cell lysates prepared from E. coli cells overexpressing WT or S88L TrmD. Cells were first grown at 30 °C for 1 h (A₆₀₀ = ∼0.2) and then shifted to 30, 37, and 43 °C, respectively, for 8 h, and cell lysates with 10 μg of total protein were loaded to a 12% SDS-PAGE and probed with an anti-TrmD antibody, followed by detection with a secondary antibody. The signal of TrmD co-migrated with a purified enzyme in a separate 12% SDS-PAGE stained by Coomassie Blue. The intensity of each signal was quantified using imaging analysis and was calculated relative to the level of each enzyme at 30 °C. The reported ratios were the average of two independent measurements. F, intracellular level of m¹G37-tRNA as determined by the resistance to RNaseH digestion. EctRNA^Leu/CAG was purified from total E. coli tRNA using a biotin-tagged oligonucleotide specific to the D-loop region. Total refers to the combined pool of both G37- and m¹G37-tRNA, whereas m¹G37 refers to the fraction of only m¹G37-tRNA in the combined pool. Transcript refers to the unmodified tRNA^Leu/CAG prepared from transcription of the tRNA gene. TS 43 °C refers to the tRNA isolated from E. coli S88L-trmD cells grown at 43 °C for 10 h, whereas TS 30 °C refers to the tRNA isolated from E. coli S88L-trmD cells grown at 30 °C for 10 h. The full-length and nuclease-cleaved fragments of the tRNA were separated on a denaturing 12% PAGE, 7 m urea, and the quantities were estimated by ethidium bromide stain.