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. 2013 Jan 28;52(5):1441-5.
doi: 10.1002/anie.201207567. Epub 2012 Nov 27.

Rewiring translation for elongation factor Tu-dependent selenocysteine incorporation

Caroline Aldag  1 , Markus J Bröcker, Michael J Hohn, Laure Prat, Gifty Hammond, Abigail Plummer, Dieter Söll
Affiliations

Rewiring translation for elongation factor Tu-dependent selenocysteine incorporation

Caroline Aldag et al. Angew Chem Int Ed Engl. .
No abstract available

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Figures

Fig. 1
Fig. 1
Secondary structure of E. coli tRNASer and tRNASec, and tRNAUTu. E. coli tRNASer is the major scaffold of tRNAUTu, the acceptor stem originates from tRNASec (blue), and recognition elements for EF-Tu were retained from tRNASer (red). The amber anticodon CUA (orange) is tRNAUTuam, while the opal anticodon UCA (green) defines tRNAUTuop.
Fig. 2
Fig. 2
(A) tRNAUTu mediates functional Sec incorporation in FDHH. An E. coli ΔselAΔselBΔfdhf deletion strain was complemented with E. coli SelA, M. jannaschii PSTK, and (A1) tRNAUTuop and FDHH op; (A4) tRNAUTuam and FDHH am. Controls lacked (A2) tRNAUTuop or (A3) tRNAUTuam, and tested FDHH op and FDHH am with E. coli tRNASec and recombinant selB. FDHH activity visualized by the purple color in the benzyl viologen assay. (B) 75Se incorporation into E. coli FDHH. The E. coli culture described in Fig. 2A4 was grown in the presence of [75Se]selenite. 6xHis-tagged FDHH protein was purified and analyzed by SDS-Page (lane 1) followed by autoradiography (lane 2); FDHH corresponds to the protein band of ~80 kDa. (C) ThyA C146U restores thymine prototrophy. An E. coli ΔselAΔselBΔthyA deletion strain was complemented with wild type thyA+, thyA146Ser or thyA146am alongside with tRNAUTuam and SelA. All clones showed growth on M9 minimal medium supplemented with thymine (C1) while only thyA146am (expressing C146U ThyA) was able to reconstitute the wild type phenotype (ThyA+) on M9 minimal medium in the absence of thymine (C2).
Fig. 3
Fig. 3
Characterization of Grx1 and GPx1 mutants. (A) Glutathione disulfide oxidoreductase activity of Grx1 variants. Pure C11U/C14S Grx1, C11S/C14S Grx1 and C11/C14S Grx1 were tested for disulfide oxidoreductase activity. NADPH consumption was followed at 340 nm as a function of Grx1 concentration. (B) Peroxidase activity of Grx1 variants. Pure C11U/C14S Grx1, C11S/C14S Grx1 and C11/C14S Grx1 were tested for peroxidase activity. NADPH consumption was monitored at 340 nm as a function of reduced glutathione concentration. (C) GPx1 peroxidase activity. Peroxidase activity of recombinant Sec containing 49U GPx1 and Cys containing 49C GPx1 was compared to GPx1hum from human erythrocytes. GPx1 activity was determined with the Sigma cellular activity assay kit. Experiments shown in figures A & C were performed in triplicate, and bars indicate the standard error of the mean.
Fig. 4
Fig. 4
Mass spectroscopic confirmation of Sec incorporation. The presence of selenocysteine at amino acid position 11 in pure C11U/C14S Grx1 was confirmed by mass spectroscopy. Shown is the MS/MS spectrum of the trypsin-digested Sec-containing fragment S9G10U11P12Y13S14V15R16. Fragments observed in the second mass spectrometric analysis of this peptide are labeled b3, y2, y3, y4 and y5. The unit m/z describes the mass-to-charge ratio.
Scheme 1
Scheme 1
Aminoacyl-tRNA formation and first steps of protein synthesis. (A) Canonical amino acids: aa-tRNA gets delivered by EF-Tu to the ribosome. (B) Selenocysteine gets formed while bound to tRNA; Sec-tRNA transfer to the ribosome and accurate codon recognition are achieved by SelB (Sec-specific elongation factor) and the SECIS element (RNA structure within the open reading frame of bacterial selenoprotein mRNAs).
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