| RRC ID |
34797
|
| Author |
Kusuba H, Yoshida T, Iwasaki E, Awai T, Kazayama A, Hirata A, Tomikawa C, Yamagami R, Hori H.
|
| Title |
In vitro dihydrouridine formation by tRNA dihydrouridine synthase from Thermus thermophilus, an extreme-thermophilic eubacterium.
|
| Journal |
J Biochem
|
| 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.
|
| Volume |
158(6)
|
| Pages |
513-21
|
| Published |
2015-12-1
|
| DOI |
10.1093/jb/mvv066
|
| PII |
mvv066
|
| PMID |
26112661
|
| MeSH |
Bacterial Proteins / chemistry*
Bacterial Proteins / isolation & purification
Enzyme Assays
Oxidation-Reduction
Oxidoreductases / chemistry*
Oxidoreductases / isolation & purification
RNA, Transfer / chemistry*
Recombinant Proteins / chemistry
Recombinant Proteins / isolation & purification
Temperature
Thermus thermophilus / enzymology*
Uridine / analogs & derivatives*
Uridine / biosynthesis
|
| IF |
2.476
|
| Times Cited |
11
|
|
WOS Category
|
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| Resource |
| DNA material |
Thermus thermophilus expression plasmid TEx17D01 (THR006873)
Thermus thermophilus HB8 gene disruption plasmid TDs01F01 (THR020521). |
