Structural asymmetry of the terminal catalytic complex in selenocysteine synthesis

Selenocysteine (Sec), the 21st amino acid, is synthesized from a serine precursor in a series of reactions that require selenocysteine tRNA (tRNA^Sec). In archaea and eukaryotes, O-phosphoseryl-tRNA^Sec:selenocysteinyl-tRNA^Sec synthase (Sep^SecS) catalyzes the terminal synthetic reaction during which the phosphoseryl intermediate is converted into the selenocysteinyl moiety while being attached to tRNA^Sec. We have previously shown that only the Sep^SecS tetramer is capable of binding to and recognizing the distinct fold of tRNA^Sec. Because only two of the four tRNA-binding sites were occupied in the crystal form, a question was raised regarding whether the observed arrangement and architecture faithfully recapitulated the physiologically relevant ribonucleoprotein complex important for selenoprotein formation. Herein, we determined the stoichiometry of the human terminal synthetic complex of selenocysteine by using small angle x-ray scattering, multi-angle light scattering, and analytical ultracentrifugation. In addition, we provided the first estimate of the ratio between Sep^SecS and tRNA^Sec in vivo. We show that Sep^SecS preferentially binds one or two tRNA^Sec molecules at a time and that the enzyme is present in large molar excess over the substrate tRNA in vivo. Moreover, we show that in a complex between Sep^SecS and two tRNAs, one enzyme homodimer plays a role of the noncatalytic unit that positions CCA ends of two tRNA^Sec molecules into the active site grooves of the other, catalytic, homodimer. Finally, our results demonstrate that the previously determined crystal structure represents the physiologically and catalytically relevant complex and suggest that allosteric regulation of Sep^SecS might play an important role in regulation of selenocysteine and selenoprotein synthesis.