Herring Stephanie, Ambrogelly Alexandre, Polycarpo Carla R, Söll Dieter
Department of Molecular Biophysics, Yale University, New Haven, CT 06520-8114, USA.
Nucleic Acids Res. 2007;35(4):1270-8. doi: 10.1093/nar/gkl1151. Epub 2007 Jan 31.
Pyrrolysine (Pyl), the 22nd co-translationally inserted amino acid, is incorporated in response to a UAG amber stop codon. Pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate tRNA, the special amber suppressor tRNA(Pyl). The genes for tRNA(Pyl) (pylT) and PylRS (pylS) are found in all members of the archaeal family Methanosarcinaceae, and in Desulfitobacterium hafniense. The activation and aminoacylation properties of D. hafniense PylRS and the nature of the tRNA(Pyl) identity elements were determined by measuring the ability of 24 mutant tRNA(Pyl) species to be aminoacylated with the pyrrolysine analog N-epsilon-cyclopentyloxycarbonyl-l-lysine. The discriminator base G73 and the first base pair (G1.C72) in the acceptor stem were found to be major identity elements. Footprinting analysis showed that PylRS binds tRNA(Pyl) predominantly along the phosphate backbone of the T-loop, the D-stem and the acceptor stem. Significant contacts with the anticodon arm were not observed. The tRNA(Pyl) structure contains the highly conserved T-loop contact U54.A58 and position 57 is conserved as a purine, but the canonical T- to D-loop contact between positions 18 and 56 was not present. Unlike most tRNAs, the tRNA(Pyl) anticodon was shown not to be important for recognition by bacterial PylRS.
吡咯赖氨酸（Pyl）是第22个共翻译插入的氨基酸，它是在遇到UAG琥珀色终止密码子时被掺入的。吡咯赖氨酰 - tRNA合成酶（PylRS）将Pyl连接到其同源tRNA，即特殊的琥珀色抑制tRNA（Pyl）上。tRNA（Pyl）（pylT）和PylRS（pylS）的基因存在于甲烷八叠球菌科古菌家族的所有成员以及嗜热栖热放线菌中。通过测量24种突变型tRNA（Pyl）物种被吡咯赖氨酸类似物N - ε - 环戊氧基羰基 - L - 赖氨酸氨酰化的能力，确定了嗜热栖热放线菌PylRS的激活和氨酰化特性以及tRNA（Pyl）识别元件的性质。发现鉴别碱基G73和受体茎中的第一个碱基对（G1.C72）是主要的识别元件。足迹分析表明，PylRS主要沿着T环、D茎和受体茎的磷酸主链与tRNA（Pyl）结合。未观察到与反密码子臂有明显接触。tRNA（Pyl）结构包含高度保守的T环接触U54.A58，并且位置57保守为嘌呤，但在位置18和56之间不存在典型的T环到D环接触。与大多数tRNA不同，tRNA（Pyl）的反密码子对于细菌PylRS的识别并不重要。
