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.
Highlights
Selenocysteine (Sec) is the 21st amino acid and defining entity for selenoproteins [1]
Because a full TrxR1 activity is dependent on the presence of a Sec residue in its C-terminal active site [12,13], we first assessed whether active recombinant TrxR1 could be expressed in E. coli utilizing constructs having either UGA, UGC or UGG codons at the position corresponding to Sec
The 75Se incorporation with UGG still required the presence of a Sec Insertion Sequence (SECIS) element in the construct, implying selenoprotein synthesis machinery-dependent Sec-mediated wobble decoding at the single UGG codon located directly upstream of the SECIS element (Figure 2C)
Summary
Selenocysteine (Sec) is the 21st amino acid and defining entity for selenoproteins [1]. The genetic code is redefined in a unique process whereby specific Sec-encoding UGA codons are diverted from their natural roles of serving as translational termination signals [4]. This redefining process requires a cis-acting secondary structure in the selenoprotein mRNA—a socalled Sec Insertion Sequence (SECIS) element [5]—that is recognized by a specialized Sec insertion machinery [6]. We found Sec-mediated wobble decoding to be possible, and, when using overproduction of the selA, selB and selC genes together with UGG decoding, it allowed for recombinant selenoprotein production in E. coli
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