The Human SepSecS-tRNA ^Sec Complex Reveals the Mechanism of Selenocysteine Formation

Abstract

Selenocysteine is the only genetically encoded amino acid in humans whose biosynthesis occurs on its cognate transfer RNA (tRNA). O-Phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS) catalyzes the final step of selenocysteine formation by a poorly understood tRNAdependent mechanism. The crystal structure of human tRNA Sec in complex with SepSecS, phosphoserine, and thiophosphate, together with in vivo and in vitro enzyme assays, supports a pyridoxal phosphate-dependent mechanism of Sec-tRNA Sec formation. Two tRNA Sec molecules, with a fold distinct from other canonical tRNAs, bind to each SepSecS tetramer through their 13-base pair acceptor-TΨC arm (where Ψ indicates pseudouridine). The tRNA binding is likely to induce a conformational change in the enzyme's active site that allows a phosphoserine covalently attached to tRNA Sec , but not free phosphoserine, to be oriented properly for the reaction to occur.The 21st amino acid, selenocysteine (Sec), is distinct from other amino acids not only because it lacks its own tRNA synthetase, but also because it is the only one that is synthesized on the cognate tRNA in all domains of life [reviewed in (1-4)] in a process that is reminiscent of the tRNA-dependent synthesis of glutamine, asparagine, and cysteine in prokaryotes (4). The importance of Sec is illustrated by the embryonic lethal phenotype of the tRNA Sec knockout mouse (5) and by the presence of Sec in the active sites of enzymes involved in removing reactive oxidative species and in thyroid hormone activation (6,7). It is intriguing that the codon for Sec is UGA, which is normally a translational stop signal (1). During translation of selenoprotein mRNAs, UGA is recoded by the interaction of a

Iodothyronine deiodinases: a functional and evolutionary perspective

“…The functional implications of this variation are unknown, as there is no clear evidence that the two forms of the SECIS element differ in their effects upon mRNA translation in other selenoproteins (Fagegaltier et al 2000). Furthermore, deiodinase transcription requires an ancient and very complex trans-acting machinery; the mechanisms involved in this process were recently reviewed (Lu & Holmgren 2009, Palioura et al 2009). …”

Leishmania donovani Encodes a Functional Selenocysteinyl-tRNA Synthase

“…The ORF of L. donovani selenocysteinyl-tRNA synthase (LdSepSecS) encodes a polypeptide of 595 amino acids. As is the case in mammals and archaea (4,8,9), LdSepSecS was also found to exist as a tetramer. Leishmania SepSecS enzyme was found to be active and able to complement ⌬selA deletion in the E. coli JS1 strain.…”

“…The N terminus mediates tetramer formation such that two SepSecS dimers further associate to form a homo-tetramer (4). The structural and mechanistic insights of SepSecS action have been deciphered in eukaryotes (4,8,9). Two mechanisms have been proposed that ensure that SepSecS does not act on free O-phospho-L-serine.…”

“…Two mechanisms have been proposed that ensure that SepSecS does not act on free O-phospho-L-serine. tRNA binding has been reported to induce a conformational change in the active site of the enzyme that does not allow reaction with free phosphoserine (9) and an active site insertion is observed to contribute to PLP binding and positions a glutamate next to PLP where it repels free O-phospho-L-serine (4). The presence of selenoprotein and selenocysteine biosynthetic pathways has been reported in trypanosomes (10 -13).…”

The Long D-stem of the Selenocysteine tRNA Provides Resilience at the Expense of Maximal Function

“…The pellets were washed with 300 l of 0.5ϫ Tris borate-EDTA containing 5 mM MgSO 4 , resuspended in 800 l of the same buffer, and transferred to a glass cuvette containing 100 l of 200 mM sodium formate and 100 l of 20 mM benzyl viologen. Both sodium formate and benzyl viologen solutions were prepared with 0.1 M potassium phosphate, pH 7.0 buffer.…”