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

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• The kinase responsible for phosphorylating seryl-tRNA to form phosphoseryl-tRNA, essential for selenocysteine insertion, remained unidentified for decades.
• Previous research identified a minor seryl-tRNA decoding UGA as selenocysteine (Sec) tRNA[Ser]Sec, but the enzymatic activity was elusive.

Purpose of the Study:

• To identify the elusive kinase activity involved in selenocysteine insertion.
• To characterize the identified kinase and its role in selenoprotein biosynthesis.

Main Methods:

• Comparative genomics approach to identify candidate kinase-like genes in archaeal genomes.
• Cloning and expression of the candidate gene (mouse pstk) to produce the protein product (PSTK).
• Biochemical characterization of PSTK activity, including substrate specificity and cofactor requirements (ATP, Mg2+).

Main Results:

• A candidate gene for mammalian phosphoseryl-tRNA[Ser]Sec kinase (pstk) was identified through comparative genomics.
• The expressed PSTK protein specifically phosphorylated the seryl moiety on seryl-tRNA[Ser]Sec, requiring ATP and Mg2+.
• Homologs of PSTK were found in archaea and eukaryotes (Drosophila, C. elegans), but not in bacteria, plants, or yeast, indicating conserved function in selenoprotein synthesis.

Conclusions:

• The identified PSTK is the missing kinase activity essential for selenocysteine insertion into tRNA.
• PSTK is highly conserved across archaea and eukaryotes, underscoring its critical role in selenoprotein biosynthesis and/or regulation.
• The absence of PSTK in bacteria, plants, and yeast suggests a specific evolutionary path for selenoprotein synthesis in these domains.