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Engineering the elongation factor Tu for efficient selenoprotein synthesis.

Haruna K, Alkazemi MH, Liu Y, Söll D, Englert M - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.

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EF-Tu variant sequences and their effect on UTu-mediated selenoprotein formation. (A) The structure (10) of the ternary complex of EF-Tu (cyan) with GTP and Cys-tRNACys (red and atomic coloring of Cys) is aligned to that (14) of SelB (brown). Residues forming the aa binding pocket are indicated using E. coli numbering. (B) hAGT UAG145 and the UTu components were supplemented with EF-Tu, EF-R1 and EF-R2 and subjected to three selection rounds before a 10−5 dilution was plated to indicate the surviving fraction. (C) Amino acid alignment of wild-type E. coli EF-Tu with the various EF-Tu variants and SelB.
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Figure 2: EF-Tu variant sequences and their effect on UTu-mediated selenoprotein formation. (A) The structure (10) of the ternary complex of EF-Tu (cyan) with GTP and Cys-tRNACys (red and atomic coloring of Cys) is aligned to that (14) of SelB (brown). Residues forming the aa binding pocket are indicated using E. coli numbering. (B) hAGT UAG145 and the UTu components were supplemented with EF-Tu, EF-R1 and EF-R2 and subjected to three selection rounds before a 10−5 dilution was plated to indicate the surviving fraction. (C) Amino acid alignment of wild-type E. coli EF-Tu with the various EF-Tu variants and SelB.

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).


Engineering the elongation factor Tu for efficient selenoprotein synthesis.

Haruna K, Alkazemi MH, Liu Y, Söll D, Englert M - Nucleic Acids Res. (2014)

EF-Tu variant sequences and their effect on UTu-mediated selenoprotein formation. (A) The structure (10) of the ternary complex of EF-Tu (cyan) with GTP and Cys-tRNACys (red and atomic coloring of Cys) is aligned to that (14) of SelB (brown). Residues forming the aa binding pocket are indicated using E. coli numbering. (B) hAGT UAG145 and the UTu components were supplemented with EF-Tu, EF-R1 and EF-R2 and subjected to three selection rounds before a 10−5 dilution was plated to indicate the surviving fraction. (C) Amino acid alignment of wild-type E. coli EF-Tu with the various EF-Tu variants and SelB.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150793&req=5

Figure 2: EF-Tu variant sequences and their effect on UTu-mediated selenoprotein formation. (A) The structure (10) of the ternary complex of EF-Tu (cyan) with GTP and Cys-tRNACys (red and atomic coloring of Cys) is aligned to that (14) of SelB (brown). Residues forming the aa binding pocket are indicated using E. coli numbering. (B) hAGT UAG145 and the UTu components were supplemented with EF-Tu, EF-R1 and EF-R2 and subjected to three selection rounds before a 10−5 dilution was plated to indicate the surviving fraction. (C) Amino acid alignment of wild-type E. coli EF-Tu with the various EF-Tu variants and SelB.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.

Show MeSH
Related in: MedlinePlus