title: Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs.
abstract: 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.
authors.forename: Debabrata
authors.initials: D
authors.lastname: Mandal
authors.forename: Caroline
authors.initials: C
authors.lastname: Köhrer
authors.forename: Dan
authors.initials: D
authors.lastname: Su
authors.forename: I Ramesh
authors.initials: IR
authors.lastname: Babu
authors.forename: Clement T Y
authors.initials: CT
authors.lastname: Chan
authors.forename: Yuchen
authors.initials: Y
authors.lastname: Liu
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
authors.forename: Paul
authors.initials: P
authors.lastname: Blum
authors.forename: Masayasu
authors.initials: M
authors.lastname: Kuwahara
authors.forename: Peter C
authors.initials: PC
authors.lastname: Dedon
authors.forename: Uttam L
authors.initials: UL
authors.lastname: Rajbhandary
mesh_terms: D000926:Anticodon / Q000235:genetics*; D020029:Base Pairing; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D019614:Haloarcula marismortui / Q000235:genetics*; D019621:Haloferax / Q000235:genetics; D015394:Molecular Structure; D017354:Point Mutation; D019642:RNA, Archaeal / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics; D012331:RNA, Fungal / Q000235:genetics; D012355:RNA, Transfer, Ile / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016944:Sulfolobus / Q000235:genetics; D046249:Transfer RNA Aminoacylation; D014529:Uridine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics
chemical_list: D000926:Anticodon; D003062:Codon; D019642:RNA, Archaeal; D012329:RNA, Bacterial; D012331:RNA, Fungal; D012355:RNA, Transfer, Ile; D014529:Uridine
keywords: 5-cyanomethyl uridine; anticodon wobble position; archaea; codon reading properties; tRNA modification