Identification and characterization of phosphoseryl-tRNA ^[Ser]Sec kinase

Abstract

In 1970, a kinase activity that phosphorylated a minor species of seryl-tRNA to form phosphoseryl-tRNA was found in rooster liver [Maenpaa, P. H. & Bernfield, M. R. (1970) Proc. Natl. Acad. Sci. USA 67, 688 -695], and a minor seryl-tRNA that decoded the nonsense UGA was detected in bovine liver. The phosphoseryl-tRNA and the minor UGA-decoding seryl-tRNA were subsequently identified as selenocysteine (Sec) tRNA [Ser]Sec , but the kinase activity remained elusive. Herein, by using a comparative genomics approach that searched completely sequenced archaeal genomes for a kinase-like protein with a pattern of occurrence similar to that of components of Sec insertion machinery, we detected a candidate gene for mammalian phosphoseryl-tRNA [ S elenocysteine (Sec) has its own code word, UGA, and its own tRNA, and therefore is viewed as the 21st amino acid in the genetic code (reviewed in refs. 1-4). Although UGA usually codes for the termination of protein synthesis, it also specifies Sec if specific requirements are met. The presence of a stemloop structure downstream of UGA, called a Sec insertion sequence (SECIS) element, is the critical component in selenoprotein mRNAs that dictates UGA to code for Sec (reviewed in ref. 5). In mammals, the SECIS element occurs in the 3Ј untranslated region of selenoprotein mRNAs. A specific elongation factor, EFsec, specifically recognizes selenocysteyltRNA [Ser]Sec (6, 7), and a SECIS element binding protein, SBP2, binds specifically to the SECIS element (8), directing the insertion of Sec into protein in response to UGA.It has been known for several years that the biosynthesis of Sec occurs on its tRNA in both bacteria (9) and mammals (10) after the tRNA is initially aminoacylated with serine by seryl-tRNA synthetase. In Escherichia coli, the pathway for the biosynthesis of Sec has been completely established (reviewed in ref. 1). After the aminoacylation of bacterial tRNA [Ser]Sec with serine, the hydroxyl group is removed from the seryl moiety to yield an aminoacrylyl intermediate, and this step is catalyzed by a pyridoxal phosphate-dependent Sec synthase. The aminoacrylyl intermediate serves as the acceptor for the activated selenium donor, monoselenophosphate, which is synthesized from selenite and ATP in the presence of selenophosphate synthetase (reviewed in ref. 1). Once selenium is donated to the intermediate, the biosynthesis of Sec on tRNA [Ser]Sec is complete.In eukaryotes, however, the biosynthesis of Sec has not been established, but several components have been identified over the years that play a role in this process. For example, in 1970, a minor seryl-tRNA was reported to form phosphoseryl-tRNA by a kinase activity from rooster liver (11), and a minor seryl-tRNA from bovine, rabbit, and chicken livers was reported to specifically decode the nonsense codon UGA (12). Although it was subsequently shown that both the minor seryl-tRNA that formed phosphoseryl-tRNA and the one that decoded UGA were the same tRNA (13), it was not known at the time these components were ...

Protein Interactome and Its Application to Protein Function Prediction

“…species), they are considered as functionally associated (Barker & Pagel, 2005;Bowers et al, 2004;Pellegrini et al, 1999). These methods applied to microorganisms and successfully discovered novel participants of known pathway (Carlson et al, 2004;Luttgen et al, 2000).…”

New Developments in Selenium Biochemistry: Selenocysteine Biosynthesis in Eukaryotes and Archaea

“…The fact that both SLA and PSTK sequences are found in eukaryotes and archaea that utilize the Sec protein insertion machinery, but not in those organisms that do not, provide strong evidence that this pathway is used by all eukaryotes and archaea that synthesize Sec-containing proteins ( [13,18] and also see [30]). …”

“…This tRNA was subsequently identified as Sec tRNA [Ser]Sec [6]. The kinase, designated O-phosphoseryl-tRNA [Ser]Sec kinase (PSTK), was recently isolated, characterized, and found to specifically phosphorylate the seryl moiety on seryl-tRNA [Ser]Sec [18]. The resulting O-phosphoseryltRNA [Ser]Sec product had been proposed as an intermediate in the biosynthesis of Sec [3,18].…”

“…We used a computational, comparative genomics approach to identify a single kinase with homologies common only to archaea and eukaryotes that utilize the Sec protein insertion machinery [18]. We also used a similar comparative genomics approach to identify SLA as the likely SecS [13].…”

“…The kinase, designated O-phosphoseryl-tRNA [Ser]Sec kinase (PSTK), was recently isolated, characterized, and found to specifically phosphorylate the seryl moiety on seryl-tRNA [Ser]Sec [18]. The resulting O-phosphoseryltRNA [Ser]Sec product had been proposed as an intermediate in the biosynthesis of Sec [3,18]. In addition, because no sequences with homology to SelA could be detected in eukaryotes, there was some question as to the identity of the Sec synthase (SecS) in mammals.…”