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. 2014 Dec 23;16(1):91–113. doi: 10.3390/ijms16010091

Figure 5.

Figure 5

T. acidophilum Trm1 (the Ta0997 gene product) is a single-site-specific enzyme. (A) The T. acidophilum Ta0997 gene product (left), T. kodakaraensis Trm1 (center) and A. aeolicus Trm1 (right) (2 µg each) were analyzed by 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue; (B) The tRNALeuUAG transcript was methylated by the Ta0997 gene product and then the generated methylated nucleotide was analyzed by 2D-TLC; (C) The sequences of S. cerevisiae tRNAPhe, A. aeolicus tRNATyr and A. aeolicus tRNATyr A26G, G27A are depicted by cloverleaf structures. These tRNA transcripts were previously used to determine the site specificity of A. aeolicus Trm1, which is a multi-site-specific Trm1. The nucleotides at position 26 in the tRNA transcripts are colored red. The transcript numbers (1, 2, and 3) correspond to the lane numbers in panels (D–F). (D) The tRNA transcripts (0.1 A260 units each) were incubated with the Ta0997 gene product (T. acidophilum Trm1) and 14C-AdoMet at 50 °C for 5 min, separated by 10% PAGE (7 M urea), and then the gel was analyzed by autoradiography. The left and right panels show the gel stained with methylene blue and its autoradiogram, respectively. T. kodakaraensis (E) and A. aeolicus (F) Trm1 proteins were analyzed by the same method.