In vitro dihydrouridine formation by tRNA dihydrouridine synthase from Thermus thermophilus, an extreme-thermophilic eubacterium
| Autor: | Akira Hirata, Takako Awai, Ai Kazayama, Chie Tomikawa, Eri Iwasaki, Hiroaki Kusuba, Ryota Yamagami, Takeshi Yoshida, Hiroyuki Hori |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Biochemistry
law.invention chemistry.chemical_compound Bacterial Proteins RNA Transfer law Eubacterium Molecular Biology Gene Uridine Enzyme Assays chemistry.chemical_classification biology Chemistry Thermophile Thermus thermophilus Temperature General Medicine biology.organism_classification Recombinant Proteins Enzyme Transfer RNA Recombinant DNA Dihydrouridine Oxidoreductases Oxidation-Reduction |
| Zdroj: | Journal of biochemistry. 158(6) |
| ISSN: | 1756-2651 |
| Popis: | Dihydrouridine (D) is formed by tRNA dihydrouridine synthases (Dus). In mesophiles, multiple Dus enzymes bring about D modifications at several positions in tRNA. The extreme-thermophilic eubacterium Thermus thermophilus, in contrast, has only one dus gene in its genome and only two D modifications (D20 and D20a) in tRNA have been identified. Until now, an in vitro assay system for eubacterial Dus has not been reported. 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 (Δdus) strain of T. thermophilus verified that the two D20 and D20a modifications in tRNA were derived from one Dus protein. The Δdus strain did not show growth retardation at any temperature. The assay system showed that Dus modified tRNA(Phe) 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 tRNA(Phe) purified from the Δdus strain and tRNA(Phe) transcript revealed that other tRNA modifications are required for D formation at high temperatures. |
| Databáze: | OpenAIRE |
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