The Sec biosynthesis pathway in archaea and eukaryotes. Sec in synthesized on tRNA^Sec in three steps. 1. The unacylated tRNA^Sec is serylated by SerRS; 2. the resulting Ser-tRNA^Sec is phosphorylated by PSTK forming Sep-tRNA^Sec; 3. the phosphorylated intermediate is converted to the final product Sec-tRNA^Sec by SepSecS. The crystal structures of tRNA and enzymes in this pathway are presented: tRNA^Sec (ribbon) from Homo sapiens [19], SerRS (ribbon) with AMP (stick) from Pyrococcus horikoshii [64], PSTK (ribbon) from M. jannaschii [26], and SepSecS (ribbon) with tRNA^Sec (stick, only one tRNA is shown) from Homo sapiens [18].

Structure and evolution of PylRS and the Pyl operon. (A) Key hydrogen-bond interactions in the active site of M. mazei PylRS with pyrrolysyl-adenylate (Pyl-AMP) bound. (B) Two views of the D. hafniense PylRS/tRNA^Pyl complex showing the tRNA binding domain 1 (cyan), the catalytic core domain (tan), the bulge domain (yellow), the C-terminal tail domain (green), and α-helix 6 (blue) emerging from the other protomer (gray). The tRNA core binding surface (magenta highlight) and U shaped concave surface (purple highlight) are also illustrated. Pyl-AMP from the M. mazei PylRS structure was superimposed onto the DhPylRS structure for reference. (C) The PylRS maximum likelihood phylogenetic tree includes all known putative Pyl-decoding organisms to date, and the organization of the Pyl operon in each genome sequence is shown adjacent to the tree. Square brackets indicate partial sequence data from a short sequence read, and curly brackets indicate that the Pyl operon is split between different contigs from a metagenomic survey. * indicates an in-frame TAG codon. Sequence data were downloaded from the Integrated Microbial Genomes database [65]. The tree was calculated with PHYML [66] using a BioNJ starting tree and SPR tree search followed by NNI branch swapping to optimize the tree. All alignment positions were considered, likelihood parameters were estimated from the alignment, and the JTT+Γ model with 8 rate categories was applied. Bootstrap values (only those > 50 are shown) were computed according to the Shimodaira-Hasegawa re-estimation of log-likelihood test implemented in PHYML.

Binding to SepSecS promotes a conformational change in tRNA^Sec. (A) The secondary structure of human tRNA^Sec with bases mentioned in the text highlighted in bold. (B) Superposition of the sugar-phosphate backbone of both the D and anticodon arms of free tRNA^Sec (blue) on the corresponding atoms of tRNA^Sec complexed with SepSecS (red) reveals a conformational change in the tRNA molecule on binding to the enzyme. The variable arm of tRNA^Sec rotates by 33°, the T arm swings 17° and the acceptor arm rotates by 6° around the axis that runs parallel to both the D and anticodon arms. The free tRNA^Sec conformer cannot bind to SepSecS because the tip of its acceptor arm would clash with the helix α1 (arrow) and the variable arm would be positioned away from the helix α9. (C) The acceptor arm also slides down towards the anticodon arm on binding to SepSecS through a 24° rotation around the axis that is parallel to the variable arm. This movement positions G73 to interact with Arg398 in the helix α14 and orients the CCA end toward the active site. Free tRNA^Sec is blue, tRNA^Sec complexed with SepSecS is red and SepSecS is beige. Only the secondary structure elements of SepSecS that interact with tRNA are shown. The view is first rotated ~30° clockwise around the horizontal axis, and then another 30° anticlockwise around the vertical axis relative to panel B.