title: Reconstitution of selenocysteine incorporation reveals intrinsic regulation by SECIS elements.
abstract: Selenoproteins are present in all three domains of life and are responsible for a major part of a cell's antioxidant defense against reactive oxygen species. Synthesis of selenoproteins requires the decoding of a UGA codon as selenocysteine (Sec) instead of translation termination. Sec is incorporated into the growing polypeptide chain during translation elongation and is known to require a set of highly specific factors: the Sec insertion sequence (SECIS) element in the 3' untranslated region, Sec-tRNA(Sec), the Sec-specific elongation factor eEFSec, and SECIS binding protein 2. Since reconstitution has not been reported, whether these factors are sufficient is unknown. Here, we report a novel in vitro translation system in which Sec incorporation has been reconstituted from purified components introduced into a Sec naive system. In addition, we developed a novel method to purify Sec-tRNA(Sec) and active eEFSec/GTP/tRNA ternary complex. We found that the known basal factors are sufficient for Sec incorporation in vitro. Using this highly manipulable system, we have also found that ribosomes from non-Sec-utilizing organisms cannot support Sec incorporation and that some SECIS elements are intrinsically less efficient than others. Having identified the essential set of factors, this work removes a significant barrier to our understanding of the mechanism of Sec incorporation.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.
authors.forename: Nirupama
authors.initials: N
authors.lastname: Gupta
authors.forename: Louise W
authors.initials: LW
authors.lastname: DeMong
authors.forename: Sowmya
authors.initials: S
authors.lastname: Banda
authors.forename: Paul R
authors.initials: PR
authors.lastname: Copeland
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D002457:Cell Extracts; D002474:Cell-Free System; D018388:Codon, Terminator; D008297:Male; D010445:Peptide Elongation Factors / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D051381:Rats; D012270:Ribosomes / Q000378:metabolism; D017279:Selenocysteine / Q000235:genetics* / Q000378:metabolism*; D013737:Testis
chemical_list: D020413:3' Untranslated Regions; D002457:Cell Extracts; D018388:Codon, Terminator; D010445:Peptide Elongation Factors; D012333:RNA, Messenger; D012345:RNA, Transfer, Amino Acid-Specific; D016601:RNA-Binding Proteins; C036928:tRNA, selenocysteine-; D017279:Selenocysteine