Figure 7.
TsaE is required for multiple turnover of the t6A biosynthesis reaction. (A) Standard radiochemical t6A assays measuring the incorporation of [14C]-threonine into tRNA. Assays contained Thermotoga maritima TsaC2 together with TsaB, TsaD and TsaE (open squares) or TsaB and TsaD (filled squares). (B) Native agarose gel-shift analysis showing the association of wild-type TsaE and mutants TsaEE108A and TsaET42A with TsaB2D2 in the absence of nucleotides. (C) TLC radiogram of the ATP hydrolysis reactions (generating ADP and Pi) catalyzed by wild-type TsaE, and mutants TsaEE108A and TsaET42A, in the absence and presence of TsaB2D2. [γ-32P] ATP and the radiolabeled hydrolysis product γ-phosphate are indicated. (D) Standard radiochemical t6A assays measuring the incorporation of [14C]-threonine into tRNA. Assays contained T. maritima TsaC2, TsaB and TsaD either alone (filled squares), or with added TsaEE108A (open squares) or TsaET42A (open triangles). (E) Radiochemical t6A assays ([14C]threonine) analyzed by native PAGE. Reaction assays were carried out as described in the ‘Materials and Methods’ section, and contained 8 μM each of TsaC2, TsaB and TsaD ([TsaB2D2] therefore 4 μM). The [tRNA] in assays analyzed in lanes 1, 3 and 5 was 40 μM, while the [tRNA] in the assay analyzed in lane 4 was 3.75 μM. Lane 1. Standard assay (assay contains TsaB, TsaD and TsaE). Lane 2. Control lane—assay lacking tRNA. Lane 3. Assay contains TsaB and TsaD. Lane 4. Assay contains TsaB and TsaD (low tRNA). Lane 5. Assay contains TsaB, TsaD and TsaEE108A. Lane 6. Control lane—[14C]-labeled t6A-modified tRNA.