Abstract
Selenocysteine (Sec), the 21(st) 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 (SepSecS) 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 SepSecS 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 SepSecS and tRNA(Sec) in vivo. We show that SepSecS 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 SepSecS 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 SepSecS might play an important role in regulation of selenocysteine and selenoprotein synthesis.