ABSTRACT
The 21st amino acid, selenocysteine (Sec), is synthesized on its cognate transfer RNA (tRNA(Sec)). In bacteria, SelA synthesizes Sec from Ser-tRNA(Sec), whereas in archaea and eukaryotes SepSecS forms Sec from phosphoserine (Sep) acylated to tRNA(Sec). We determined the crystal structures of Aquifex aeolicus SelA complexes, which revealed a ring-shaped homodecamer that binds 10 tRNA(Sec) molecules, each interacting with four SelA subunits. The SelA N-terminal domain binds the tRNA(Sec)-specific D-arm structure, thereby discriminating Ser-tRNA(Sec) from Ser-tRNA(Ser). A large cleft is created between two subunits and accommodates the 3'-terminal region of Ser-tRNA(Sec). The SelA structures together with in vivo and in vitro enzyme assays show decamerization to be essential for SelA function. SelA catalyzes pyridoxal 5'-phosphate-dependent Sec formation involving Arg residues nonhomologous to those in SepSecS. Different protein architecture and substrate coordination of the bacterial enzyme provide structural evidence for independent evolution of the two Sec synthesis systems present in nature.
Subject(s)
Bacteria/enzymology , Bacterial Proteins/chemistry , RNA, Transfer, Amino Acyl/chemistry , Selenocysteine/biosynthesis , Transferases/chemistry , Arginine/chemistry , Catalysis , Catalytic Domain , Crystallography, X-Ray , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Pyridoxal Phosphate/chemistrySubject(s)
Peptide Elongation Factor Tu/metabolism , Selenocysteine/metabolism , Formate Dehydrogenases/chemistry , Formate Dehydrogenases/genetics , Formate Dehydrogenases/metabolism , Glutathione Peroxidase/chemistry , Glutathione Peroxidase/genetics , Glutathione Peroxidase/metabolism , Humans , Hydrogenase/chemistry , Hydrogenase/genetics , Hydrogenase/metabolism , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Peptide Elongation Factor Tu/chemistry , Protein Biosynthesis , Protein Engineering , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Ribosomes/metabolismABSTRACT
Robust molecular markers such as microsatellites are important tools used to understand the dynamics of natural populations, but their identification and development are typically time consuming and labor intensive. The recent emergence of so-called next-generation sequencing raised the question as to whether this new technology might be applied to microsatellite development. Following this view, we considered whether deep sequencing using the 454 Life Sciences/Roche GS-FLX genome sequencing system could lead to a rapid protocol to develop microsatellite primers as markers for genetic studies. For this purpose, genomic DNA was sourced from three unrelated organisms: a fungus (the pine pathogen Fusarium circinatum), an insect (the pine-damaging wasp Sirex noctilio), and the wasp's associated nematode parasite (Deladenus siricidicola). Two methods, FIASCO (fast isolation by AFLP of sequences containing repeats) and ISSR-PCR (inter-simple sequence repeat PCR), were used to generate microsatellite-enriched DNA for the 454 libraries. From the resulting 1.2-1.7 megabases of DNA sequence data, we were able to identify 873 microsatellites that have sufficient flanking sequence available for primer design and potential amplification. This approach to microsatellite discovery was substantially more rapid, effective, and economical than other methods, and this study has shown that pyrosequencing provides an outstanding new technology that can be applied to this purpose.
