Fig. 1: Identification and biochemical characterization of Up at position 47 of S. tokodaii tRNAVal3.
From: Reversible RNA phosphorylation stabilizes tRNA for cellular thermotolerance
a, Secondary structure of S. tokodaii tRNAVal3 with post-transcriptional modifications. N324 is shown in red. The cleavage sites of RNase T1 and RNase A that generate RNA fragments with N324 are indicated by black and grey triangles, respectively. b, CID spectrum of the N324-containing fragment (m/z 852.59) of S. tokodaii tRNAVal2/3 digested with RNase T1. c- and y-series product ions are indicated. c, CID spectrum of the N324-containing fragment (m/z 1,215.15) of S. tokodaii tRNAVal2/3 digested with RNase A. c- and y-series product ions are indicated. d, RNA-MS of RNA fragments with or without N324 and Um50. The upper panel shows the base peak chromatogram (BPC) of RNase T1-digested fragments. The second, third, fourth and fifth panels represent extracted ion chromatograms (XICs) of the divalent negative ions of the respective fragments, as indicated. e, Chemical structure of 2′-phosphouridine (Up). The 2′-phosphate group is shown in red. f, Melting curves of S. tokodaii tRNAVal3 with (red line) or without (blue line) Up47. Tm values were determined on the basis of the inflection point of the melting curve. Data represent the average values of technical triplicates ± s.d. P < 1.02 × 10–4 by two-sided Student’s t test. g, RNase probing of S. tokodaii tRNAVal3 with (red line) or without (blue line) Up47. Top, PAGE gels showing degradation of 32P-labelled tRNA with or without Up47 by RNase I at the indicated time (min). Intact tRNA is indicated by a triangle. Data represent the average values of technical triplicates ± s.d. The unprocessed gel image is provided in Supplementary Fig. 10.
