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. 2017 Mar;23(3):406–419. doi: 10.1261/rna.059667.116

FIGURE 1.

FIGURE 1.

The tRNAThr(CGU)28,50 scaffold used for analysis of tRNAThr variants is fully functional and efficiently modified to m3C. (A) Schematic of tRNAThr(CGU)28,50. The secondary structure of tRNAThr(CGU) is shown. The base pairs of residues 28:42 and 50:64 are switched as indicated in the boxes. The primer complementary to residues 55–36 is indicated with a 3′ arrow. (B) tRNAThr(CGU)28,50 is functional in vivo. tT(CGU)Δ cells containing the integrated WT tT(CGU) or tT(CGU)28,50 as indicated were grown overnight in YPD medium, serially diluted and spotted onto YPD medium, and plates were incubated at indicated temperatures for 3 d. (C) tRNAThr(CGU)28,50 is efficiently modified to m3C32. Bulk RNA from cells containing the integrated WT tT(CGU) or tT(CGU)28,50 as indicated were analyzed by primer extension assay, as described in Materials and Methods, using the primer shown in A. The vast majority of tRNAThr(CGU)28,50 has m3C32 based on the primer extension stop at U33, compared to the amount of read-through (RT).