A sequence in the Escherichia coli fdhF “selenocysteine insertion sequence” (SECIS) operates in the absence of selenium

Liu, Zesheng; Reches, Myriam; Engelberg‐Kulka, Hanna

doi:10.1006/jmbi.1999.3307

Cited by 21 publications

(11 citation statements)

References 40 publications

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“…However, the corresponding gst-ЈprxUЈ-ЈlacZ fusion did not show appreciable ␤-galactosidase activity in the absence of SelB and tRNA Sec from E. acidaminophilum, whereas synthesis of full-length PrxU from the vector pASK-IBA3 revealed strong UGA suppression. Thus, this high level of selenium-independent readthrough cannot be explained solely by the features of the sequence surrounding the selenocysteine codon (27,33,34). Other factors, such as codon usage, growth conditions, and the expression system itself, might play an important role (31,35).…”

Section: Discussionmentioning

confidence: 99%

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

Gursinsky

Gröbe

Schierhorn

et al. 2008

Appl Environ Microbiol

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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 grampositive 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.The 21st amino acid selenocysteine (Sec) is present in a variety of different eukaryotic, archaeal, and bacterial proteins (11,25,26,29,50). The insertion of selenocysteine into growing peptide chains is directed by an opal (UGA) codon (8, 51), which in the standard genetic code signals termination of translation. Selenoprotein synthesis in Escherichia coli has been elucidated and intensively studied by Böck and coworkers (6) and requires the products of the genes selA, selB, selC, and selD. selC encodes a selenocysteine-specific tRNA Sec that is first charged with serine by seryl-tRNA synthetase. It is converted to selenocysteyl-tRNA Sec by selenocysteine synthase, the gene product of selA. A low-molecular-weight selenium donor, selenophosphate, is required for this reaction. It is synthesized from ATP and selenide by selenophosphate synthetase, SelD (41).Recoding of UGA into selenocysteine requires a special mRNA motif, the selenocysteine insertion sequence (SECIS) (22). In mRNAs encoding bacterial selenoproteins, it is located at the 3Ј site of the UGA codon (52). This mRNA structure is recognized by the bacterial selenocysteine-specific elongation factor SelB, which functions homologously to the standard elongation factor EF-Tu (9, 24). SelB binds GTP and selenocysteyl-...

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Section: Discussionmentioning

confidence: 99%

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

Gursinsky

Gröbe

Schierhorn

et al. 2008

Appl Environ Microbiol

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“…Single colonies were inoculated in 2 to 5 ml of Luria broth (LB) medium and incubated at 37°C overnight with vigorous shaking; 50 l or 1 ml of the overnight cultures was added to 5 ml or 100 ml of LB at 25 or 37°C as indicated. In all expression experiments 5 M Na 2 SeO 3 was added to ensure sufficient selenium supply and suppress Trp incorporation at the UGA (25) and 100 g of L-Cys per ml was included to prevent unspecific incorporation of selenium into proteins through sulfur pathways (29). The cultures were grown with shaking until the optical density at 600 nm (OD 600 ) reached 0.50 to 0.55.…”

Section: Methodsmentioning

confidence: 99%

Assessment of Production Conditions for Efficient Use ofEscherichia coliin High-Yield Heterologous Recombinant Selenoprotein Synthesis

Rengby

Johansson

Carlson

et al. 2004

Appl Environ Microbiol

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The production of heterologous selenoproteins in Escherichia coli necessitates the design of a secondary structure in the mRNA forming a selenocysteine insertion sequence (SECIS) element compatible with SelB, the elongation factor for selenocysteine insertion at a predefined UGA codon. SelB competes with release factor 2 (RF2) catalyzing translational termination at UGA. Stoichiometry between mRNA, the SelB elongation factor, and RF2 is thereby important, whereas other expression conditions affecting the yield of recombinant selenoproteins have been poorly assessed. Here we expressed the rat selenoprotein thioredoxin reductase, with titrated levels of the selenoprotein mRNA under diverse growth conditions, with or without cotransformation of the accessory bacterial selA, selB, and selC genes. Titration of the selenoprotein mRNA with a pBAD promoter was performed in both TOP10 and BW27783 cells, which unexpectedly could not improve yield or specific activity compared to that achieved in our prior studies. Guided by principal component analysis, we instead discovered that the most efficient bacterial selenoprotein production conditions were obtained with the high-transcription T7lac-driven pET vector system in presence of the selA, selB, and selC genes, with induction of production at late exponential phase. About 40 mg of rat thioredoxin reductase with 50% selenocysteine content could thereby be produced per liter bacterial culture. These findings clearly illustrate the ability of E. coli to upregulate the selenocysteine incorporation machinery on demand and that this is furthermore strongly augmented in late exponential phase. This study also demonstrates that E. coli can indeed be utilized as cell factories for highly efficient production of heterologous selenoproteins such as rat thioredoxin reductase.Many organisms express selenoproteins, carrying a selenocysteine residue, the 21st naturally occurring amino acid (7,16,26,34). Selenocysteine is cotranslationally inserted at the position of an opal (UGA) codon, which normally confers termination of translation. The UGA codon is recoded as selenocysteine by complex translation machineries that differ between gram-negative (7, 16) and gram-positive bacteria (13), archaea (31), and higher eukaryotes (9,12,30). The translation system in Escherichia coli is the most characterized (reviewed in references 7, 16, and 34). Briefly, the mRNA for an E. coli selenoprotein carries a specific sequence after the UGA codon, both encoding the amino acids following the selenocysteine residue and forming a stem-loop secondary structure, a so-called selenocysteine insertion sequence (SECIS) element.The SECIS element binds the SelB elongation factor, the selB gene product. SelB is homologous to elongation factor Tu (EF-Tu) but, in addition, binds the loop of the SECIS element through an additional C-terminal domain. In terms of tRNA substrate, SelB is only functional with the selenocysteine-specific tRNA Sec , the selC gene product, in its selenocysteinylated form. By analogy with ...

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“…All recombinant DNA manipulations were carried out by standard procedures (24). Site-directed mutagenesis was carried out as we have described previously (18,19). Restriction enzymes and other enzymes used in the recombinant DNA experiments were obtained from New England Biolabs (Beverly, Mass.).…”

Section: Methodsmentioning

confidence: 99%

The Bulged Nucleotide in the Escherichia coli Minimal Selenocysteine Insertion Sequence Participates in Interaction with SelB: a Genetic Approach

Li¹,

Reches²,

Engelberg‐Kulka³

2000

J Bacteriol

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The UGA codon, which usually acts as a stop codon, can also direct the incorporation into a protein of the amino acid selenocysteine. This UGA decoding process requires a cis-acting mRNA element called the selenocysteine insertion sequence (SECIS), which can form a stem-loop structure. In Escherichia coli, selenocysteine incorporation requires only the 17-nucleotide-long upper stem-loop structure of the fdhF SECIS. This structure carries a bulged nucleotide U at position 17. Here we asked whether the single bulged nucleotide located in the upper stem-loop structure of the E. coli fdhF SECIS is involved in the in vivo interaction with SelB. We used a genetic approach, generating and characterizing selB mutations that suppress mutations of the bulged nucleotide in the SECIS. All the selB suppressor mutations isolated were clustered in a region corresponding to 28 amino acids in the SelB C-terminal subdomain 4b. These selB suppressor mutations were also found to suppress mutations in either the loop or the upper stem of the E. coli SECIS. Thus, the E. coli SECIS upper stem-loop structure can be considered a "single suppressible unit," suggesting that there is some flexibility to the nature of the interaction between this element and SelB.The UGA codon, which usually acts as a stop codon, can also direct incorporation of the amino acid selenocysteine (for reviews, see references 4, 5, 7, and 25). This UGA decoding process requires a cis-acting mRNA element called the selenocysteine insertion sequence (SECIS), which can form a stemloop structure (4, 9, 15; for reviews, see references 2 and 20).In Escherichia coli, a number of genes have been identified in which the UGA directs the incorporation of selenocysteine. These include genes fdhF (27) and fdnG (3), encoding the selenocysteine-containing enzymes formate dehydrogenase H and N, respectively. Immediately downstream from the selenocysteine-specifying UGA in the mRNA of each of these polypeptides is found a SECIS that has been described as consisting of at least 40 nucleotides capable of forming a stem-loop RNA structure (2, 9). In later work, it was suggested that an extended fdhF SECIS was required, consisting of an additional helix of 7 bp in which the U and G residues of the UGA codon are included and the A residue is bulged out (10). After carrying out an extensive mutational analysis of the fdhF SECIS DNA, we found that for in vivo UGA-directed selenocysteine incorporation, there is no requirement for the whole stem-loop RNA structure of the E. coli fdhF SECIS (including the extended form) (18), as thought previously. Instead, the 17-bp upper stem-loop structure is sufficient to permit selenocysteine incorporation on the condition that it is located 11 nucleotides downstream from the UGA codon (Fig. 1). This mini upper stem-loop structure contains a bulged nucleotide, a U residue, located 17 nucleotides downstream from the UGA (Fig. 1). Selenocysteine incorporation into an fdhF-lacZЈ fusion polypeptide depends on both (i) the specificity of nucleotide 17 as a...

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A sequence in the Escherichia coli fdhF “selenocysteine insertion sequence” (SECIS) operates in the absence of selenium

Cited by 21 publications

References 40 publications

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

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

Assessment of Production Conditions for Efficient Use ofEscherichia coliin High-Yield Heterologous Recombinant Selenoprotein Synthesis

The Bulged Nucleotide in the Escherichia coli Minimal Selenocysteine Insertion Sequence Participates in Interaction with SelB: a Genetic Approach

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