Fukunaga Ryuya, Harada Yoko, Hirao Ichiro, Yokoyama Shigeyuki
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Biochem Biophys Res Commun. 2008 Aug 1;372(3):480-5. doi: 10.1016/j.bbrc.2008.05.078. Epub 2008 May 27.
An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) could expand the genetic alphabet and allow the incorporation of non-standard amino acids into proteins at defined positions. For this purpose, we synthesized tRNAs bearing Pa at the anticodon and tested non-standard amino acid phosphoserine aminoacylation by the wild-type and various engineered phosphoseryl-tRNA synthetases (SepRSs). The D418N D420N T423V triple mutant of SepRS efficiently charged phosphoserine to tRNA containing the PaUA anticodon with a K(m)=47.1muM and a k(cat)=0.151s(-1), which are comparable to the values of the wild-type SepRS for its cognate substrate, tRNA(Cys) with the GCA anticodon (26.9muM and 0.111s(-1)). The triple mutant SepRS and the tRNA with the PaUA anticodon represent a specific pair for the site-specific incorporation of phosphoserine into proteins in response to the UADs codon within mRNA.
7-(2-噻吩基)-咪唑并[4,5-b]吡啶（Ds）与吡咯-2-甲醛（Pa）之间的非天然碱基对能够扩展遗传字母表，并允许在特定位置将非标准氨基酸掺入蛋白质中。为此，我们合成了反密码子处带有Pa的tRNA，并测试了野生型和各种工程化的磷酸丝氨酰-tRNA合成酶（SepRSs）对非标准氨基酸磷酸丝氨酸的氨酰化作用。SepRS的D418N D420N T423V三重突变体能够有效地将磷酸丝氨酸加载到含有PaUA反密码子的tRNA上，其K(m)=47.1μM，k(cat)=0.151s(-1)，这与野生型SepRS对其同源底物（带有GCA反密码子的tRNA(Cys)）的值（26.9μM和0.111s(-1)）相当。三重突变体SepRS和带有PaUA反密码子的tRNA代表了一对特定组合，可用于在mRNA中的UADs密码子响应下将磷酸丝氨酸位点特异性掺入蛋白质中。
