SecS proteins are shown in red; SelA in pink, and archaeal SelA-like proteins in green. Other PLP-dependent protein branches are compressed and represented by family names. The phylogenetic tree was generated by ClustalW and PHYLIP programs. Both bootstrap support (the number of times each branch was supported in bootstrap replication) and the measurement of distance for the branch lengths (shown by a bar) are indicated.

GenBank accession numbers for the sequences are given in the Accession Numbers summary. The active site lysine (K), which is PLP-binding residue, is indicated with an arrow.

(A) Relative efficiencies of tRNA^[Ser]Sec, seryl-tRNA^[Ser]Sec, and O-phosphoseryl-tRNA^[Ser]Sec binding to mSecS are shown. Cloning of mSecS-Cys, its expression and purification, binding assays, and Northern analysis are detailed in Materials and Methods. The upper panel shows the binding of each form of tRNA^[Ser]Sec to mSecS-Cys. The lower panel shows the amount of each tRNA^[Ser]Sec used in the binding reaction which was assessed by Northern blot analysis of 2 μl of each binding reaction solution designated as tRNA loading control.
(B) Amounts of each tRNA^[Ser]Sec form bound to mSecS in (A) above were quantified by measuring the band densities of each form bound to mSecS relative to those of the corresponding densities of the amount of each form added to the assay using ImageQuant Version 5.2 (Molecular Dynamics). The error bars were derived from four separate binding assays. The p-value in each case is <0.01.
(C) ³²P-labeled O-phosphoseryl-tRNA^[Ser]Sec was added to a reaction mixture containing either mSecS, SelA, mSPS2-Cys, or SelD, and the reaction was incubated, the aminoacyl-tRNA^[Ser]Sec was deacylated, and the deacylated products were chromatographed as given in Materials and Methods. Lane 1 contains ³²P-labeled O-phosphoserine, and the other lanes contain the components shown.

Cloning of the genes, mouse sps1, mouse sps2, SelD, and C. elegans sps2, and mutation of mouse sps2 to sps2-Cys and reaction conditions are detailed in Materials and Methods. For NMR analysis, 200 μl of ATP hydrolysis reaction was sealed in 3-mm NMR tubes and incubated at 37 °C for 4 h before ³¹P NMR spectroscopic analysis [9].
(A) Complete ³¹P-NMR spectra of ATP hydrolysis products generated with mSPS2-Cys, C. elegans selenophosphate synthetase 2, SelD, and mSPS1 are shown.
(B) Expanded spectra of the ordinate and abscissa between 15 and 30 ppm for these products are shown.
(C) ATP hydrolysis reactions with [α-³²P]ATP, either without or with 0.25 mM selenide, incubated with mSPS2-Cys, C. elegans selenophosphate synthetase 2, SelD, or mSPS1; at the end of the incubation period, reactions were loaded onto PEI TLC plates, run in 0.8 M LiCl, and exposed to a PhosphorImager screen as described in Materials and Methods.

All reactions were carried out under anaerobic conditions and are detailed in Materials and Methods. Synthetic tRNA^[Ser]Sec was used in those reactions employing tRNA and its synthesis, aminoacylation with ³H-serine and phosphorylation were carried out as given [20]. Cloning of mouse sps2, and preparation of the Sec-to-Cys sps2 mutant, and cloning of E. coli SelA and mouse SecS are given in Materials and Methods.
(A) Sec biosynthesis using SeP as the active selenium donor with O-phosphoseryl-tRNA^[Ser]Sec or seryl-tRNA^[Ser]Sec and either mSecS or SelA as SecS is shown.
(B) Sec biosynthesis using mSPS2-Cys and selenide (HSe⁻) to provide SeP as the active selenium donor with O-phosphoseryl-tRNA^[Ser]Sec or seryl-tRNA^[Ser]Sec and either mSecS or SelA as SecS is shown. HSe⁻ was maintained in the reduced state in reactions in B with DTT as described in Materials and Methods. Migration of control amino acids and pyruvate are indicated below the graphs in (A) and (B).
(C) The rate of Sec synthesis is shown. Reactions were terminated at 0, 1.25, 2.5, 5, 10, 20, 40, and 80 min. After chromatography and counting of samples in a liquid scintillation counter as given in Materials and Methods, the counts from the peaks of Sec, O-phosphoserine, or the degraded intermediate (the peak migrated after alanine and chromatographed with pyruvate) were pooled together for quantification at each time point. The analyses in this figure were carried out on deacylated products.

The pathway of Sec biosynthesis is shown (see text for details and abbreviations are defined in the text with the exception of SerS [seryl-tRNA synthetase] and PSTK [O-phosphoseryl-tRNA^[Ser]Sec kinase]).