Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea
Hfx. volcanii is insensitive to GC7 and spermidine is the substrate of T. kodakarensis DHS. (a) Effect of GC7 on the growth of H26. Hfx. volcanii H26 (WT, parent) was diluted to OD600 of 1 and then spot-plated (15 μL) on solid agar Hv_min medium in serial dilutions as indicated in absence or in presence of 1 mM GC7. Each experiment was performed with two biological replicates and three technical replicates. (b) Detection of T. kodakarensis aIF5A (lanes 2, 3, 4, 6, 7, and 8) and S. cerevisiae eIF5A (lanes 1 and 5) modification using [14C] spermidine or [3H] putrescine as substrates in presence of T. kodakarensis DHS (lanes 3 and 7) or S. cerevisiae DHS (lanes 1, 2, and 5). The in vitro assay was resolved on a 16.5% tricine polyacrylamide gel. The dried gels were exposed to autoradiography films to visualize possible modifications. The assembly of each reaction is depicted above. (i), Coomassie Blue staining; (ii), exposed autoradiography film; (iii), overlay of the Coomassie Blue stained gel and the exposed film; indicates the presence of the radioactivity; molecular mass markers are indicated in kDa; T.k, Thermococcus kodakarensis; S.c, Saccharomyces cerevisiae.
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