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Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

Journal Article · · RNA
 [1];  [2];  [3];  [3];  [3];  [4];  [4];  [5];  [6];  [3];  [2]
  1. Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Dept. of Biology; DOE/OSTI
  2. Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Dept. of Biology
  3. Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Dept. of Biological Engineering
  4. Yale Univ., New Haven, CT (United States). Dept. of Molecular Biophysics and Biochemistry
  5. Univ. of Nebraska, Lincoln, NE (United States). School of Biological Sciences
  6. Gunma Univ. (Japan). Graduate School of Science and Technology
+ Show Author Affiliations
Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA2 Ile) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA2 Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.
Research Organization:
Univ. of Nebraska, Lincoln, NE (United States)
Sponsoring Organization:
USDOE Office of Energy Efficiency and Renewable Energy (EERE)
Grant/Contract Number:
FG36-08GO88055
OSTI ID:
1627096
Journal Information:
RNA, Journal Name: RNA Journal Issue: 2 Vol. 20; ISSN 1355-8382
Publisher:
Cambridge University PressCopyright Statement
Country of Publication:
United States
Language:
English

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Related Subjects

5-cyanomethyl uridine
59 BASIC BIOLOGICAL SCIENCES
Biochemistry & Molecular Biology
anticodon wobble position
archaea
codon reading properties
tRNA modification