RNA-dependent selenocysteine biosynthesis in eukaryotes and Archaea
This is an Open Access Thesis
Selenocysteine (Sec), the 21st genetically encoded amino acid, is the major metabolite of the micronutrient selenium. Sec is inserted into nascent proteins in response to a UGA codon. The substrate for ribosomal protein synthesis is selenocysteinyl-tRNASec. While the formation of Sec-tRNASec from seryl-tRNASec by a single bacterial enzyme selenocysteine synthase (SelA) has been well described, the mechanism of Sec-tRNASec formation in archaea and eukaryotes remained poorly understood. Herein, biochemical and genetic data provide evidence that, in contrast to bacteria, eukaryotes and archaea utilize a different route to Sec-tRNASec that requires the tRNASec-dependent conversion of O-phosphoserine (Sep) to Sec. In this two-step pathway, O-phosphoseryl-tRNA kinase (PSTK) first converts Ser-tRNASec to Sep-tRNASec. This misacylated tRNA is the obligatory precursor for a Sep-tRNA: Sec-tRNA synthase (SepSecS); this protein was previously annotated as Soluble Liver Antigen/Liver Pancreas (SLA/LP). SepSecS genes from Homo sapiens, the lower eukaryote Trypanosoma brucei and the archaea Methanocaldococcus jannaschii and Methanococcus maripaludis complement an Escherichia coli DeltaselA deletion strain in vivo. Furthermore, genetic analysis of selenoprotein biosynthesis in T. brucei in vivo demonstrated that eukaryotes have a single pathway to Sec-tRNASec that requires Sep-tRNASec as an intermediate. Finally, purified recombinant SepSecS converts Sep-tRNA Sec into Sec-tRNASec in vitro in the presence of sodium selenite and purified E. coli selenophosphate synthetase.The final step in Sec biosynthesis was further investigated by a structure-based mutational analysis of the M. maripaludis SepSecS and by determining the crystal structure of human SepSecS complexed with tRNA Sec, phosphoserine and thiophosphate at 2.8 A resolution. In vivo and in vitro enzyme assays support a mechanism of Sec-tRNASec formation based on pyridoxal phosphate, while the lack of active site cysteines demonstrates that a perselenide intermediate is not involved in SepSecS-catalyzed Sec formation. Two tRNASec molecules, with a fold distinct from other canonical tRNAs, bind to each human SepSecS tetramer through their unique 13 base-pair acceptor-TPsiC arm. The tRNA binding induces a conformational change in the enzyme's active site that allows a Sep covalently attached to tRNASec, but not free Sep, to be oriented properly for the reaction to occur.