Engineering the elongation factor Tu for efficient selenoprotein synthesis.
Bottom Line: Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis.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.
Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.Show MeSH
Related in: MedlinePlus
Mentions: The crystal structure (10) of the ternary complex of Thermus aquaticus EF-Tu, E. coli Cys-tRNACys and GTP (PDB 1B23) was our guide for EF-Tu engineering (Figure 2). Residues near the aminoacyl moiety attached to the 3′ end of the tRNA make up the aa binding pocket of EF-Tu. Sec-tRNASec is delivered by its own elongation factor, SelB, whose structure is known (PDB 4ACB) (14). A structural alignment of EF-Tu with SelB indicates a similar fold of both aa binding pockets, albeit with variations in residues forming a pronounced negatively charged surface on EF-Tu and, conversely, a positively charged surface on SelB (Figure 2). The alignment of several EF-Tu and SelB protein sequences highlights these differences: H67Y, E216D, D217R and N274R (using EF-Tu numbering).
Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.