A tRNA-dependent cysteine biosynthesis enzyme recognizes the selenocysteine-specific tRNA in Escherichia coli

Abstract

The essential methanogen enzyme Sep-tRNA:Cys-tRNA synthase (SepCysS) converts O-phosphoseryl-tRNA(Cys) (Sep-tRNA(Cys)) into Cys-tRNA(Cys) in the presence of a sulfur donor. Likewise, Sep-tRNA:Sec-tRNA synthase converts O-phosphoseryl-tRNA(Sec) (Sep-tRNA(Sec)) to selenocysteinyl-tRNA(Sec) (Sec-tRNA(Sec)) using a selenium donor. While the Sep moiety of the aminoacyl-tRNA substrates is the same in both reactions, tRNA(Cys) and tRNA(Sec) differ greatly in sequence and structure. In an Escherichia coli genetic approach that tests for formate dehydrogenase activity in the absence of selenium donor we show that Sep-tRNA(Sec) is a substrate for SepCysS. Since Sec and Cys are the only active site amino acids known to sustain FDH activity, we conclude that SepCysS converts Sep-tRNA(Sec) to Cys-tRNA(Sec), and that Sep is crucial for SepCysS recognition.

Fig. 1. Secondary structures of tRNA^Sec and tRNA^Cys

The cloverleaf structures of E. coli tRNA^Sec (A) and tRNA^Cys (B) are shown.

Fig. 2. SepCysS and PSTK complement an E. coli ΔselA deletion

The indicated proteins (middle) complement the loss of selenocysteine synthase (SelA) in the E. coli ΔselA deletion strain JS1. Activity of the selenoproteins FDH_H and FDH_N was tested with the benzyl viologen assay (left) and the McConkey nitrate plate assay (right) respectively.

Fig. 3. Metabolic labeling of transformed ΔselA strains with ⁷⁵Se

The E. coli ΔselA strain JS1 was complemented with E. coli selA (lane 1), empty vector control (lane 2), M. jannaschii spcS (coding for SepSecS, lane 3), M. jannaschii pscS (coding for SepCysS, lane 4), M. jannaschii spcS and pstK genes (lane 5), and M. jannaschii pscS and pstK (lane 6). Two major bands were observed in the positive control lane 1. Based on the molecular weight marker, the upper band corresponds to FDH_H. The lower band is likely a degradation product of FDH_H, the sole selenoprotein in E. coli in the indicated growth conditions.

Fig. 4. SepCysS restores FDH_H activity in an E. coli ΔselA ΔselD strain

The indicated proteins (right) complement the loss of SelA and SelD in the E. coli strain MH1. Activity of the selenoprotein FDH_H is tested with benzyl viologen assay.