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Factors and selenocysteine insertion sequence requirements for the synthesis of selenoproteins from a gram-positive anaerobe in Escherichia coli.

Abstract：

: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 (75)Se 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 (tRNA(Sec)) 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 (75)Se-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.

journal_name

Appl Environ Microbiol

journal_title

Applied and environmental microbiology

authors

Gursinsky T,Gröbe D,Schierhorn A,Jäger J,Andreesen JR,Söhling B

doi

10.1128/AEM.02238-07

subject

Has Abstract

pub_date

2008-03-01 00:00:00

pages

1385-93

issue

eissn

0099-2240

issn

1098-5336

pii

AEM.02238-07

journal_volume

pub_type

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Factors and selenocysteine insertion sequence requirements for the synthesis of selenoproteins from a gram-positive anaerobe in Escherichia coli.