The DXD motif of Trf4p is crucial for its poly(A) polymerase activity and the degradation of tRNAiMet. (A) Sctrf4-236 complements the cold sensitivity of trf4Δ. A strain bearing chromosomal trf4Δ was transformed with YCplac33, ScTRF4 (B254), or Sctrf4-236 (B316), and the transformants were grown on SC-URA at 30°C, then replica-printed onto SC-URA plates and incubated at 16° and 26°C for 3 d and 1 d, respectively. (B) trf4-236 suppresses the Ts− phenotype of a trm6-504 mutant. A trm6-504 trf4Δ strain (Y301) was transformed with YCpLac33, ScTRF4 (B254), and Sctrf4-236 (B316); wild type (Y200) and trm6-504 (Y190) were transformed just with YCpLac33. Individual transformants were grown to saturation in SC-URA and 10-fold serial dilutions were spotted on SC-URA plates and incubated at 26°, 36°, and 16°C for 2, 3, and 4 d, respectively. (C) Northern hybridization of total RNA (5 μg) from wild type (Y200), trm6-504(Y190), trm6-504 trf4Δ (sup1) with YCpLac33, or Sctrf4-236 strains. Total RNA (5 μg) was separated on a 6% polyacrylamide (19:1) 8 Murea gel and transferred to a membrane and probed with JA11 to detect tRNAiMet and JA 151 to detect tRNALeu. The tRNAs were visualized by autoradiography and the quantities were determined by phosphorimage analysis using a STORM 640 and ImageQuant software (GE Healthcare). The quantity of tRNAiMet in each sample is shown as a percentage of tRNAiMet in the wild-type strain after normalization with tRNACAALeu. This experiment has been repeated and the quantity of tRNAiMet was found to be similar each time. (D) The DXD motif is important for its poly(A) polymerase activity in vivo. Total RNA (5 μg) and poly(A)+ RNA (2–4 μg) isolated from the transformants described in B were separated, and a Northern hybridization was conducted to detect tRNAiMet using a radiolabeled oligonucleotide (JA11) and autoradiography.
