FIG. 6.

HPLC fractionation of methyl-esterified tRNA nucleosides from mutant yeast cells deficient in products potentially found in a complex with Trm9 (17). Total tRNA was extracted from in vivo-radiolabeled cells with mutations in the F8 (YDR140w), YNR050c, and YOL124c genes as well as in the TRM9 gene as described in the legend to Fig. 2. Subsequently, tRNA was digested with P1 nuclease and subjected to alkaline phosphatase to generate free nucleosides, which were fractionated by HPLC. Methyl ester radioactivity was measured by adding 100 μl of 2 N NaOH to 100 μl of each fraction and measuring vapor phase [³H]methanol as described in Materials and Methods. The elution positions of mcm⁵U and mcm⁵s²U methyl esters are shown with arrows. For clarity, the baseline has been adjusted; there is no significant radioactivity seen except in the region of mcm⁵U and mcm⁵s²U.