title: In vitro dihydrouridine formation by tRNA dihydrouridine synthase from Thermus thermophilus, an extreme-thermophilic eubacterium.
abstract: 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.
authors.affiliation: Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Kusuba
authors.affiliation: Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yoshida
authors.affiliation: Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Iwasaki
authors.affiliation: Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Takako
authors.initials: T
authors.lastname: Awai
authors.affiliation: Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Ai
authors.initials: A
authors.lastname: Kazayama
authors.affiliation: Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Hirata
authors.affiliation: Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Chie
authors.initials: C
authors.lastname: Tomikawa
authors.affiliation: Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Ryota
authors.initials: R
authors.lastname: Yamagami
authors.affiliation: Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan hori@eng.ehime-u.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Hori
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D057075:Enzyme Assays; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D012343:RNA, Transfer / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology*; D014529:Uridine / Q000031:analogs & derivatives* / Q000096:biosynthesis
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; C010922:5,6-dihydrouridine; D012343:RNA, Transfer; D010088:Oxidoreductases; C462500:tRNA-dihydrouridine synthase; D014529:Uridine
keywords: Dus; RNA modification; tRNA; tRNA dihydrouridine synthase; thermophiles