Thyer Ross, Robotham Scott A, Brodbelt Jennifer S, Ellington Andrew D
Institute for Cellular and Molecular Biology, University of Texas at Austin , Austin, Texas 78712, United States.
J Am Chem Soc. 2015 Jan 14;137(1):46-9. doi: 10.1021/ja510695g. Epub 2014 Dec 23.
Bacterial selenocysteine incorporation occurs in response to opal stop codons and is dependent on the presence of a selenocysteine insertion sequence (SECIS) element, which recruits the selenocysteine specific elongation factor and tRNA(Sec) needed to reassign the UGA codon. The SECIS element is a stem-loop RNA structure immediately following the UGA codon and forms part of the coding sequence in bacterial selenoproteins. Although the site specific incorporation of selenocysteine is of great interest for protein engineering, the sequence constraints imposed by the adjoining SECIS element severely limit its use. We have evolved an E. coli tRNA(Sec) that is compatible with the canonical translation machinery and can suppress amber stop codons to incorporate selenocysteine with high efficiency. This evolved tRNA(Sec) allows the production of new recombinant selenoproteins containing structural motifs such as selenyl-sulfhydryl and diselenide bonds.
细菌硒代半胱氨酸的掺入是对乳白终止密码子的响应，并且依赖于硒代半胱氨酸插入序列（SECIS）元件的存在，该元件招募重新分配UGA密码子所需的硒代半胱氨酸特异性延伸因子和tRNA（Sec）。SECIS元件是紧接在UGA密码子之后的茎环RNA结构，并且构成细菌硒蛋白编码序列的一部分。尽管硒代半胱氨酸的位点特异性掺入对于蛋白质工程非常有意义，但相邻的SECIS元件所施加的序列限制严重限制了其使用。我们已经进化出一种与经典翻译机制兼容的大肠杆菌tRNA（Sec），它可以抑制琥珀终止密码子，从而高效地掺入硒代半胱氨酸。这种进化的tRNA（Sec）允许生产含有硒代巯基和二硒键等结构基序的新型重组硒蛋白。
