作为吡咯赖氨酸-tRNA合成酶底物的吡咯赖氨酸类似物。
Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase.
作者信息
Polycarpo Carla R, Herring Stephanie, Bérubé Amélie, Wood John L, Söll Dieter, Ambrogelly Alexandre
机构信息
Department of Chemistry, Yale University, New Haven, CT 06520-8114, USA.
出版信息
FEBS Lett. 2006 Dec 11;580(28-29):6695-700. doi: 10.1016/j.febslet.2006.11.028. Epub 2006 Nov 20.
In certain methanogenic archaea a new amino acid, pyrrolysine (Pyl), is inserted at in-frame UAG codons in the mRNAs of some methyltransferases. Pyl is directly acylated onto a suppressor tRNA(Pyl) by pyrrolysyl-tRNA synthetase (PylRS). Due to the lack of a readily available Pyl source, we looked for structural analogues that could be aminoacylated by PylRS onto tRNA(Pyl). We report here the in vitro aminoacylation of tRNA(Pyl) by PylRS with two Pyl analogues: N-epsilon-d-prolyl-l-lysine (d-prolyl-lysine) and N-epsilon-cyclopentyloxycarbonyl-l-lysine (Cyc). Escherichia coli, transformed with the tRNA(Pyl) and PylRS genes, suppressed a lacZ amber mutant dependent on the presence of d-prolyl-lysine or Cyc in the medium, implying that the E. coli translation machinery is able to use Cyc-tRNA(Pyl) and d-prolyl-lysine-tRNA(Pyl) as substrates during protein synthesis. Furthermore, the formation of active beta-galactosidase shows that a specialized mRNA motif is not essential for stop-codon recoding, unlike for selenocysteine incorporation.
在某些产甲烷古菌中，一种新的氨基酸——吡咯赖氨酸（Pyl），被插入到一些甲基转移酶mRNA的框内UAG密码子处。吡咯赖氨酸-tRNA合成酶（PylRS）将Pyl直接酰化到抑制性tRNA（Pyl）上。由于缺乏现成的Pyl来源，我们寻找能够被PylRS氨基酰化到tRNA（Pyl）上的结构类似物。我们在此报告了PylRS用两种Pyl类似物对tRNA（Pyl）进行的体外氨基酰化反应：N-ε-d-脯氨酰-L-赖氨酸（d-脯氨酰赖氨酸）和N-ε-环戊氧基羰基-L-赖氨酸（Cyc）。用tRNA（Pyl）和PylRS基因转化的大肠杆菌，在培养基中存在d-脯氨酰赖氨酸或Cyc的情况下抑制了lacZ琥珀突变体，这意味着大肠杆菌的翻译机制在蛋白质合成过程中能够将Cyc-tRNA（Pyl）和d-脯氨酰赖氨酸-tRNA（Pyl）用作底物。此外，活性β-半乳糖苷酶的形成表明，与硒代半胱氨酸的掺入不同，一个专门的mRNA基序对于终止密码子重新编码不是必需的。
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