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Applied and Environmental Microbiology, March 2008, p. 1385-1393, Vol. 74, No. 5
0099-2240/08/$08.00+0     doi:10.1128/AEM.02238-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Factors and Selenocysteine Insertion Sequence Requirements for the Synthesis of Selenoproteins from a Gram-Positive Anaerobe in Escherichia coli{triangledown}

Torsten Gursinsky,{dagger} Daniel Gröbe,{ddagger} Angelika Schierhorn,§ Jana Jäger, Jan R. Andreesen, and Brigitte Söhling*

Institut für Mikrobiologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle, Germany

Received 23 September 2007/ Accepted 21 December 2007

Selenoprotein synthesis in Escherichia coli strictly depends on the presence of a specific selenocysteine insertion sequence (SECIS) following the selenocysteine-encoding UGA codon of the respective mRNA. It is recognized by the selenocysteine-specific elongation factor SelB, leading to cotranslational insertion of selenocysteine into the nascent polypeptide chain. The synthesis of three different selenoproteins from the gram-positive anaerobe Eubacterium acidaminophilum in E. coli was studied. Incorporation of 75Se into glycine reductase protein B (GrdB1), the peroxiredoxin PrxU, and selenophosphate synthetase (SelD1) was negligible in an E. coli wild-type strain and was fully absent in an E. coli SelB mutant. Selenoprotein synthesis, however, was strongly increased if selB and selC (tRNASec) from E. acidaminophilum were coexpressed. Putative secondary structures downstream of the UGA codons did not show any sequence similarity to each other or to the E. coli SECIS element. However, mutations in these structures strongly reduced the amount of 75Se-labeled protein, indicating that they indeed act as SECIS elements. UGA readthrough mediated by the three different SECIS elements was further analyzed using gst-lacZ translational fusions. In the presence of selB and selC from E. acidaminophilum, UGA readthrough was 36 to 64% compared to the respective cysteine-encoding UGC variant. UGA readthrough of SECIS elements present in Desulfomicrobium baculatum (hydV), Treponema denticola (selD), and Campylobacter jejuni (selW-like gene) was also considerably enhanced in the presence of E. acidaminophilum selB and selC. This indicates recognition of these SECIS elements and might open new perspectives for heterologous selenoprotein synthesis in E. coli.

* Corresponding author. Present address: Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle, Germany. Phone: 49-345-5524886. Fax: 49-345-5527013. E-mail: brigitte.soehling{at}biochemtech.uni-halle.de

{triangledown} Published ahead of print on 28 December 2007.

{dagger} Present address: Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle, Germany.

{ddagger} Present address: Institut für Biochemie und Molekularbiologie, Charité Universitätsmedizin-Campus Benjamin Franklin, Arnimallee 22, 14195 Berlin-Dahlem, Germany.

§ Present address: Max Planck Forschungsstelle für Enzymologie der Proteinfaltung, Weinbergweg 22, 06120 Halle, Germany.

Applied and Environmental Microbiology, March 2008, p. 1385-1393, Vol. 74, No. 5
0099-2240/08/$08.00+0     doi:10.1128/AEM.02238-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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