Fig. 5: KptA acts as a potential eraser for Up47.
From: Reversible RNA phosphorylation stabilizes tRNA for cellular thermotolerance
a, Reversibility of Up47 mediated by ArkI and KptA. ArkI phosphorylates U47 of tRNA to form Up47 using ATP as a phosphate donor, producing ADP as a by-product. KptA converts Up47 to U47 by transferring the phosphate group of Up47 to NAD+, producing ADP-ribose 1′′,2′′-cyclic phosphate (Appr>p) and nicotinamide (NA) as by-products. b, In vitro dephosphorylation of Up47 with (+) or without (–) TkKptA. XICs show the sum of monovalent and divalent negative ions from RNase T1-digested fragments containing Up47 (upper panels) or U47 (lower panels). c, Kinetic measurement of in vitro Up47 dephosphorylation by TkKptA. The initial velocity (Vi) of the dephosphorylation reaction was measured at the indicated tRNA concentrations. Data represent the average values of technical triplicates ± s.d. The Km and Vmax values are shown above the graph. d, Dephosphorylation of Up47 by TkKptA in E. coli. XICs show Up47-containing fragments derived from various E. coli tRNA species (Supplementary Table 5) from an E. coli ΔtrmBΔtapT strain expressing N. viennensis ArkI: UpCGp (top panels), UpCUGp (second panels), UpCAGp (third panels), UpCACAGp (fourth panels) and m5UΨCGp as a control fragment (bottom panels). Relative abundance of the Up47-containing fragments was measured in E. coli strains in which TkKptA was not expressed (left panels) or where TkKptA expression was induced with 10 μM (middle panels) or 100 μM (right panels) IPTG. e, Relative peak intensity of each Up47-containing fragment detected in the tRNA fraction from E. coli strains cultured with 0, 10 or 100 μM IPTG. Data represent the average values of technical triplicates ± s.d.
