Abstract
Selenocysteine (Sec) is naturally co-translationally incorporated into proteins by recoding the UGA opal codon with a specialized elongation factor (SelB in bacteria) and an RNA structural signal (SECIS element). We have recently developed a SECIS-free selenoprotein synthesis system that site-specifically—using the UAG amber codon—inserts Sec depending on the elongation factor Tu (EF-Tu). Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis. A Sec-specific selection system was established by expression of human protein O6-alkylguanine-DNA alkyltransferase (hAGT), in which the active site cysteine codon has been replaced by the UAG amber codon. The formed hAGT selenoprotein repairs the DNA damage caused by the methylating agent N-methyl-N′-nitro-N-nitrosoguanidine, and thereby enables Escherichia coli to grow in the presence of this mutagen. An EF-Tu library was created in which codons specifying the amino acid binding pocket were randomized. Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library. The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production.
Highlights
Ribosomal protein synthesis translates the information encoded on the mRNA into a nascent polypeptide according to the universal genetic code
Bacteria employ a two-step mechanism for translational incorporation of Sec: (i) the tRNASec is misacylated with serine by the endogenous seryl-tRNA synthetase (SerRS), and (ii) the serine moiety of SertRNASec is converted to Sec by selenocysteine synthase (SelA) with selenophosphate [2]
As Sec-tRNASec is not recognized by the universal elongation factor Tu (EF-Tu), the specific elongation factor SelB discriminates against the misacylated Ser-tRNASec and binds Sec-tRNASec
Summary
Ribosomal protein synthesis translates the information encoded on the mRNA into a nascent polypeptide according to the universal genetic code. Bacteria employ a two-step mechanism for translational incorporation of Sec: (i) the tRNASec is misacylated with serine by the endogenous seryl-tRNA synthetase (SerRS), and (ii) the serine moiety of SertRNASec is converted to Sec by selenocysteine synthase (SelA) with selenophosphate [2]. Natural selenoprotein synthesis proceeds through cotranslational insertion of Sec in response to a UGA opal codon. An RNA structural signal, the selenocysteine insertion sequence (SECIS) encoded immediately downstream of the UGA opal codon in bacteria, is recognized by SelB. Through ternary complex formation of Sec-tRNASec, SelB and SECIS-containing mRNA can a UGA opal codon be read unambiguously as Sec during ribosomal protein synthesis [5]. A synthetic amber suppressor tRNAUTu (a tRNA for Sec [U] that is recognized by EF-Tu) was designed to be a substrate for SerRS, SelA and EF-Tu [6]
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