title: Structural asymmetry of the terminal catalytic complex in selenocysteine synthesis.
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.
authors.affiliation: From the Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607 and.
authors.forename: Rachel L
authors.initials: RL
authors.lastname: French
authors.affiliation: the Department of Biochemistry and Molecular Biology, Rutgers-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
authors.forename: Nirupama
authors.initials: N
authors.lastname: Gupta
authors.affiliation: the Department of Biochemistry and Molecular Biology, Rutgers-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Copeland
authors.affiliation: From the Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607 and msimon5@uic.edu.
authors.forename: Miljan
authors.initials: M
authors.lastname: Simonović
mesh_terms: D000495:Allosteric Site; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry*; D020134:Catalytic Domain; D004058:Diffusion; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008027:Light; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D012313:RNA / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry*; D012542:Scattering, Radiation; D017279:Selenocysteine / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry; D014461:Ultracentrifugation; D014961:X-Ray Diffraction
chemical_list: D017279:Selenocysteine; D012313:RNA; D014364:Tryptophan; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; C542370:O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase, human
keywords: Analytical Ultracentrifugation; RNA Binding Protein; RNA-Protein Interaction; Selenocysteine; Small Angle X-ray Scattering (SAXS); Transfer RNA (tRNA)