In vitro dihydrouridine formation by tRNA dihydrouridine synthase from Thermus thermophilus, an extreme-thermophilic eubacterium

In this study, therefore, we constructed an in vitro assay system using purified Dus. Recombinant T. thermophilus Dus lacking bound tRNA was successfully purified. The in vitro assay revealed that no other factors in living cells were required for D formation. A dus gene disruptant (dus) strain of T. thermophilus verified that the two D20 and D20a modifications in tRNA were derived from one Dus protein. The dus strain did not show growth retardation at any temperature. The assay system showed that Dus modified tRNAPhe transcript at 60°C, demonstrating that other modifications in tRNA are not essential for Dus activity. However, a comparison of the formation of D in native tRNAPhe purified from the dus strain and tRNAPhe transcript revealed that other tRNA modifications are required for D formation at high temperatures.

Source: Journal of Biochemistry - November 25, 2015 Category: Biochemistry Authors: Kusuba, H., Yoshida, T., Iwasaki, E., Awai, T., Kazayama, A., Hirata, A., Tomikawa, C., Yamagami, R., Hori, H. Tags: Regular Papers Source Type: research