Factors and Selenocysteine Insertion Sequence Requirements for the Synthesis of Selenoproteins from a Gram-Positive Anaerobe in Escherichia coli
| Autor: | Jana Jäger, Angelika Schierhorn, Brigitte Söhling, Daniel Gröbe, Torsten Gursinsky, Jan R. Andreesen |
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| Rok vydání: | 2008 |
| Předmět: |
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Western Molecular Sequence Data Genetics and Molecular Biology Biology medicine.disease_cause Applied Microbiology and Biotechnology Mass Spectrometry chemistry.chemical_compound Bacterial Proteins Escherichia coli medicine RNA Messenger Insertion sequence Selenoproteins Base Pairing SECIS element Genetics chemistry.chemical_classification Messenger RNA Base Sequence Ecology Selenocysteine Eubacterium RNA Transfer Amino Acid-Specific Elongation factor chemistry Biochemistry Mutation Transfer RNA Codon Terminator Selenoprotein Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 74:1385-1393 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.02238-07 |
| Popis: | 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 . |
| Databáze: | OpenAIRE |
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