title: Wobble decoding by the Escherichia coli selenocysteine insertion machinery.
abstract: Selenoprotein expression in Escherichia coli redefines specific single UGA codons from translational termination to selenocysteine (Sec) insertion. This process requires the presence of a Sec Insertion Sequence (SECIS) in the mRNA, which forms a secondary structure that binds a unique Sec-specific elongation factor that catalyzes Sec insertion at the predefined UGA instead of release factor 2-mediated termination. During overproduction of recombinant selenoproteins, this process nonetheless typically results in expression of UGA-truncated products together with the production of recombinant selenoproteins. Here, we found that premature termination can be fully avoided through a SECIS-dependent Sec-mediated suppression of UGG, thereby yielding either tryptophan or Sec insertion without detectable premature truncation. The yield of recombinant selenoprotein produced with this method approached that obtained with a classical UGA codon for Sec insertion. Sec-mediated suppression of UGG thus provides a novel method for selenoprotein production, as here demonstrated with rat thioredoxin reductase. The results also reveal that the E. coli selenoprotein synthesis machinery has the inherent capability to promote wobble decoding.
authors: Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm SE-171 77, Sweden and Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Proteomics Karolinska (PK/KI), Karolinska Institutet, Stockholm SE-171 77, Sweden.
authors: Jianqiang
authors: J
authors: Xu
authors: Victor
authors: V
authors: Croitoru
authors: Dorothea
authors: D
authors: Rutishauser
authors: Qing
authors: Q
authors: Cheng
authors: Elias S J
authors: ES
authors: Arnér
mesh_terms: D000818:Animals; D000926:Anticodon; D003062:Codon; D004926:Escherichia coli / Q000235:genetics*; D012345:RNA, Transfer, Amino Acid-Specific / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D017279:Selenocysteine / Q000378:metabolism*; D051140:Selenoproteins / Q000096:biosynthesis* / Q000235:genetics; D054481:Thioredoxin Reductase 1 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
chemical_list: D000926:Anticodon; D003062:Codon; D012345:RNA, Transfer, Amino Acid-Specific; D011994:Recombinant Proteins; D051140:Selenoproteins; C036928:tRNA, selenocysteine-; D017279:Selenocysteine; D054481:Thioredoxin Reductase 1; C516126:Txnrd1 protein, rat
references: Angew Chem Int Ed Engl. 2013 Jan 28;52(5):1441-5
references: 23193031
references: Biochem Pharmacol. 2012 Mar 15;83(6):815-20
references: 22226931
references: EMBO J. 1992 Oct;11(10):3759-66
references: 1396569
references: Biochim Biophys Acta. 2009 Nov;1790(11):1501-12
references: 19406205
references: Antioxid Redox Signal. 2013 May 1;18(13):1675-89
references: 23121622
references: Nat Methods. 2004 Oct;1(1):61-6
references: 15782154
references: Mol Microbiol. 1991 Mar;5(3):515-20
references: 1828528
references: EMBO J. 2002 Dec 16;21(24):6925-34
references: 12486013
references: EMBO J. 1999 Apr 15;18(8):2284-93
references: 10205181
references: PLoS One. 2012;7(8):e42151
references: 22870292
references: J Biol Chem. 2004 Jan 23;279(4):2583-92
references: 14604985
references: Trends Biochem Sci. 1991 Dec;16(12):463-7
references: 1838215
references: PLoS One. 2008 Apr 02;3(4):e1846
references: 18382651
references: J Biol Chem. 1991 Apr 5;266(10):6324-8
references: 2007585
references: Methods Enzymol. 2002;347:226-35
references: 11898411
references: J Mol Biol. 2007 Feb 9;366(1):1-13
references: 17187822
references: Nucleic Acids Res. 2000 Feb 1;28(3):755-61
references: 10637327
references: J Nucl Med. 2012 Sep;53(9):1446-53
references: 22872744
references: FEBS Lett. 2013 Apr 2;587(7):906-11
references: 23428847
references: Curr Protoc Toxicol. 2001 May;Chapter 7:Unit 7.4.
references: 20954152
references: PLoS Med. 2007 Jun;4(6):e206
references: 17579510
references: J Biol Chem. 2009 Feb 6;284(6):3998-4008
references: 19054767
references: Free Radic Biol Med. 2001 Jan 1;30(1):51-61
references: 11134895
references: Appl Environ Microbiol. 2007 Jan;73(2):432-41
references: 17085697
references: Nature. 1988 Feb 25;331(6158):723-5
references: 2963963
references: Science. 2013 Apr 5;340(6128):75-8
references: 23559248
references: Biochim Biophys Acta. 2009 Jun;1790(6):495-526
references: 19364476
references: EMBO J. 2001 Dec 17;20(24):7284-93
references: 11743004
references: J Biol Chem. 2003 May 2;278(18):15966-72
references: 12574159
references: Annu Rev Nutr. 2003;23:17-40
references: 12524431
references: Free Radic Biol Med. 2009 Apr 1;46(7):893-904
references: 19146949
references: J Am Chem Soc. 2013 Jan 9;135(1):2-5
references: 23256865
references: J Mol Biol. 1999 Oct 8;292(5):1003-16
references: 10512699
references: Genes Dev. 1994 Feb 1;8(3):376-85
references: 8314089
references: Biochim Biophys Acta. 2009 Nov;1790(11):1520-32
references: 19344749
references: Appl Environ Microbiol. 2004 Sep;70(9):5159-67
references: 15345395
references: Nucleic Acids Res. 1994 Sep 11;22(18):3753-9
references: 7937088
references: Exp Cell Res. 2010 May 1;316(8):1296-303
references: 20206159
references: Nucleic Acids Res. 1981 Feb 25;9(4):983-91
references: 7015288
references: Free Radic Biol Med. 2011 Mar 15;50(6):689-99
references: 21172426
references: Mol Microbiol. 1996 Sep;21(6):1253-9
references: 8898393