RNA. 2014 Feb;20(2):177-88. doi: 10.1261/rna.042358.113. Epub 2013 Dec 16.
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.
大多数古菌和细菌在异亮氨酸 tRNA 的反密码子摆动位置使用修饰的 C 与 A 而不是 G 配对，但与 mRNA 配对。这允许 tRNA 读取异亮氨酸密码子 AUA 而不读取甲硫氨酸密码子 AUG。为了理解为什么使用修饰的 C 而不是 U 或修饰的 U 与 A 配对，我们将 Haloarcula marismortui 异亮氨酸 tRNA（tRNA2（Ile））的反密码子中的 C34 突变为 U，在 Haloferax volcanii 中表达突变 tRNA，并纯化和分析 tRNA。核糖体结合实验表明，尽管野生型 tRNA2（Ile）仅专一地与异亮氨酸密码子 AUA 结合，但突变 tRNA 不仅与 AUA 结合，还与另一个异亮氨酸密码子 AUU 结合，以及甲硫氨酸密码子 AUG。H. marismortui 另一种异亮氨酸 tRNA 物种的反密码子中的 G34 到 U 突变显示出类似的密码子结合特性。突变 tRNA 与 AUG 的结合可能导致 AUG 密码子的错误读取，并插入异亮氨酸代替甲硫氨酸。这一结果可以解释为什么大多数古菌和细菌通常不在异亮氨酸 tRNA 的反密码子摆动位置使用 U 或修饰的 U 来读取密码子 AUA。对突变 tRNA 的生化和质谱分析导致发现了一种新的修饰核苷，即突变 tRNA 反密码子摆动位置的 5-氰甲基 U。5-氰甲基 U 存在于真细菌中，但不存在于古细菌、真核生物或原核生物中。
