Selenocysteine, the 21st amino acid, has been found in 25 human selenoproteins and selenoenzymes important for fundamental cellular processes ranging from selenium homeostasis maintenance to the regulation of the overall metabolic rate. In all organisms that contain selenocysteine, both the synthesis of selenocysteine and its incorporation into a selenoprotein requires an elaborate synthetic and translational apparatus, which does not resemble the canonical enzymatic system employed for the 20 standard amino acids. In humans, three synthetic enzymes, a specialized elongation factor, an accessory protein factor, two catabolic enzymes, a tRNA, and a stem-loop structure in the selenoprotein mRNA are critical for ensuring that only selenocysteine is attached to selenocysteine tRNA and that only selenocysteine is inserted into the nascent polypeptide in response to a context-dependent UGA codon. The abnormal selenium homeostasis and mutations in selenoprotein genes have been causatively linked to a variety of human diseases, which, in turn, sparked a renewed interest in utilizing selenium as the dietary supplement to either prevent or remedy pathologic conditions. In contrast, the importance of the components of the selenocysteine-synthetic machinery for human health is less clear. Emerging evidence suggests that enzymes responsible for selenocysteine formation and decoding the selenocysteine UGA codon, which by extension are critical for synthesis of the entire selenoproteome, are essential for the development and health of the human organism.

The twenty first amino acid, selenocysteine (Sec), is the only amino acid that is synthesized on its cognate transfer RNA (tRNASec) in all domains of life. The multistep pathway involves O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS), an enzyme that catalyzes the terminal chemical reaction during which the phosphoseryl–tRNASec intermediate is converted into selenocysteinyl-tRNASec. The SepSecS architecture and the mode of tRNASec recognition have been recently determined at atomic resolution. The crystal structure provided valuable insights that gave rise to mechanistic proposals that could not be validated because of the lack of appropriate molecular probes. To further improve our understanding of the mechanism of the biosynthesis of selenocysteine in general and the mechanism of SepSecS in particular, stable tRNASec substrates carrying aminoacyl moieties that mimic particular reaction intermediates are needed. Here, we report on the accurate synthesis of methylated, phosphorylated, and phosphonated serinyl-derived tRNASec mimics that contain a hydrolysis-resistant ribose 3′-amide linkage instead of the natural ester bond. The procedures introduced allow for efficient site-specific methylation and/or phosphorylation directly on the solid support utilized in the automated RNA synthesis. For the preparation of (S)-2-amino-4-phosphonobutyric acid–oligoribonucleotide conjugates, a separate solid support was generated. Furthermore, we developed a three-strand enzymatic ligation protocol to obtain the corresponding full-length tRNASec derivatives. Finally, we developed an electrophoretic mobility shift assay (EMSA) for rapid, qualitative characterization of the SepSecS-tRNA interactions. The novel tRNASec mimics are promising candidates for further elucidation of the biosynthesis of selenocysteine by X-ray crystallography and other biochemical approaches, and could be attractive for similar studies on other tRNA-dependent enzymes.

Selenocysteine is the only amino acid that is synthesized on its cognate transfer RNA, in all domains of life. Its biosynthesis involves the enzyme O‐phosphoseryl‐tRNA: selenocysteinyl‐tRNA synthase. To explore the enzyme mechanism, tRNASec substrates carrying aminoacyl moieties that mimic reaction intermediates are needed. The cover illustrates the synthetic path of phosphonated amino acid units to the 3′‐amide linked adenosine conjugates, subsequent assembly of RNA strands and enzymatic ligation to full‐length tRNASec derivatives. The cover also symbolizes the collaborative effort of the work from the two labs involved, R. Micura and co‐workers in Innsbruck (Austria) and M. Simonović and co‐workers in Chicago (USA). For more information see the Full Paper on