title: The synthesis of methylated, phosphorylated, and phosphonated 3'-aminoacyl-tRNA(Sec) mimics.
abstract: The twenty first amino acid, selenocysteine (Sec), is the only amino acid that is synthesized on its cognate transfer RNA (tRNA(Sec)) in all domains of life. The multistep pathway involves O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS), an enzyme that catalyzes the terminal chemical reaction during which the phosphoseryl-tRNA(Sec) intermediate is converted into selenocysteinyl-tRNA(Sec). The SepSecS architecture and the mode of tRNA(Sec) recognition have been recently determined at atomic resolution. The crystal structure provided valuable insights that gave rise to mechanistic proposals that could not be validated because of the lack of appropriate molecular probes. To further improve our understanding of the mechanism of the biosynthesis of selenocysteine in general and the mechanism of SepSecS in particular, stable tRNA(Sec) substrates carrying aminoacyl moieties that mimic particular reaction intermediates are needed. Here, we report on the accurate synthesis of methylated, phosphorylated, and phosphonated serinyl-derived tRNA(Sec) mimics that contain a hydrolysis-resistant ribose 3'-amide linkage instead of the natural ester bond. The procedures introduced allow for efficient site-specific methylation and/or phosphorylation directly on the solid support utilized in the automated RNA synthesis. For the preparation of (S)-2-amino-4-phosphonobutyric acid-oligoribonucleotide conjugates, a separate solid support was generated. Furthermore, we developed a three-strand enzymatic ligation protocol to obtain the corresponding full-length tRNA(Sec) derivatives. Finally, we developed an electrophoretic mobility shift assay (EMSA) for rapid, qualitative characterization of the SepSecS-tRNA interactions. The novel tRNA(Sec) mimics are promising candidates for further elucidation of the biosynthesis of selenocysteine by X-ray crystallography and other biochemical approaches, and could be attractive for similar studies on other tRNA-dependent enzymes.
authors.affiliation: Institute of Organic Chemistry and Center for Molecular Biosciences (CMBI), Leopold-Franzens University, Center for Chemistry and Biomedicine, Innrain 80-82, 6020 Innsbruck (Austria), Fax: (+43) 512 507 57799.
authors.forename: Lukas
authors.initials: L
authors.lastname: Rigger
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Schmidt
authors.forename: Kaitlyn M
authors.initials: KM
authors.lastname: Holman
authors.forename: Miljan
authors.initials: M
authors.lastname: Simonović
authors.forename: Ronald
authors.initials: R
authors.lastname: Micura
mesh_terms: D000613:Aminobutyrates / Q000737:chemistry; D001483:Base Sequence; D001672:Biocompatible Materials / Q000138:chemical synthesis* / Q000737:chemistry; D008745:Methylation; D009690:Nucleic Acid Conformation; D010757:Phosphorous Acids / Q000737:chemistry*; D010766:Phosphorylation; D012345:RNA, Transfer, Amino Acid-Specific / Q000138:chemical synthesis / Q000737:chemistry*; D060327:Solid-Phase Synthesis Techniques
chemical_list: D000613:Aminobutyrates; D001672:Biocompatible Materials; D010757:Phosphorous Acids; D012345:RNA, Transfer, Amino Acid-Specific; C036928:tRNA, selenocysteine-; C570063:phosphonic acid; C012729:2-amino-4-phosphonobutyric acid
keywords: RNA; RNA structures; amino acids; bioconjugation; solid-phase synthesis