RNA-dependent conversion of phosphoserine forms selenocysteine in eukaryotes and archaea

Abstract

The trace element selenium is found in proteins as selenocysteine (Sec), the 21st amino acid to participate in ribosome-mediated translation. The substrate for ribosomal protein synthesis is selenocysteinyl-tRNA(Sec). Its biosynthesis from seryl-tRNA(Sec) has been established for bacteria, but the mechanism of conversion from Ser-tRNA(Sec) remained unresolved for archaea and eukarya. Here, we provide evidence for a different route present in these domains of life that requires the tRNA(Sec)-dependent conversion of O-phosphoserine (Sep) to Sec. In this two-step pathway, O-phosphoseryl-tRNA(Sec) kinase (PSTK) converts Ser-tRNA(Sec) to Sep-tRNA(Sec). This misacylated tRNA is the obligatory precursor for a Sep-tRNA:Sec-tRNA synthase (SepSecS); this protein was previously annotated as SLA/LP. The human and archaeal SepSecS genes complement in vivo an Escherichia coli Sec synthase (SelA) deletion strain. Furthermore, purified recombinant SepSecS converts Sep-tRNA(Sec) into Sec-tRNA(Sec) in vitro in the presence of sodium selenite and purified recombinant E. coli selenophosphate synthetase (SelD). Phylogenetic arguments suggest that Sec decoding was present in the last universal common ancestor. SepSecS and PSTK coevolved with the archaeal and eukaryotic lineages, but the history of PSTK is marked by several horizontal gene transfer events, including transfer to non-Sec-decoding Cyanobacteria and fungi.

Fig. 1.

tRNA-dependent amino acid transformations leading to Sec and cysteine. (Upper) The SelA route is the bacterial pathway (6). The PSTK/SepSecS is the archaeal/eukaryal route. (Lower) The SepRS/Sep-tRNA:Cys-tRNA synthase (SepCysS) pathway operates in methanogens to synthesize cysteine (16).

Fig. 2.

SepSecS genes restore FDH_H activity in an E. coli selA deletion strain. The E. coli JS1 strains complemented with the indicated SepSecS genes were grown anaerobically on glucose minimal medium plates supplemented with 0.01 mM IPTG at 30°C for 2 days. FDH_H activity was observed by overlaying top agar containing formate and BV. Colonies with active FDH_H reduced BV to a blue color. The SepSecS genes were from Methanocaldococcus jannaschii (MJ), Methanococcus maripaludis (MMP), human (HS), and E. coli selA (EC). K284T denotes a mutant human SepSecS where the Lys residue critical to PLP binding is changed to Thr.

Fig. 3.

⁷⁵Se incorporation into the E. coli selenoprotein FDH_H. Cells were grown in the presence of [⁷⁵Se]selenite in TGYEP medium (see Materials and Methods) supplemented with 0.05 mM IPTG anaerobically at 37°C for 24 h. Cell extracts were separated by 10% SDS/PAGE, and the formation of ⁷⁵Se-containing FDH_H was followed by autoradiography. The E. coli strain JS1 was complemented with E. coli SelA (lane 1), empty plasmids (lane 2), Methanocaldococcus jannaschii SepSecS (lane 3), Methanocaldococcus jannaschii SepSecS with PSTK (lane 4), and Methanocaldococcus jannaschii SepSecS, PSTK with Methanococcus maripaludis tRNA^Sec (lane 5).

Fig. 4.

Conversion of in vitro-synthesized Sep-tRNA^Sec to Sec-tRNA^Sec. Phosphorimages of TLC separation of [¹⁴C]Sep and [¹⁴C]Sec recovered from the aa-tRNAs of the SepSecS activity assay (see Materials and Methods). Sec was analyzed in its oxidized form as selenocysteic acid (Secya). Lane 1, Ser marker; lane 2, Sep marker; lane 3, Sep-tRNA^Sec with Methanococcus maripaludis SepSecS; lane 4, Sep-tRNA^Sec with human SepSecS; lane 5, Ser-tRNA^Sec with Methanococcus maripaludis SepSecS; lane 6, reaction of lane 3 with selenite omitted.

Fig. 5.

Phylogenies of SepSecS (A) and PSTK (B). Organism names are color-coded according to the domain of life: Eukarya (green), Archaea (blue), and Bacteria (red). Non-Sec-decoding organisms are labeled in bold. Scale bar shows 0.1 changes per site. Only bootstrap values <90 are shown.