Recoding the genetic code with selenocysteine

Abstract

Selenocysteine (Sec) is naturally incorporated into proteins by recoding the stop codon UGA. Sec is not hardwired to UGA, as the Sec insertion machinery was found to be able to site-specifically incorporate Sec directed by 58 of the 64 codons. For 15 sense codons, complete conversion of the codon meaning from canonical amino acid (AA) to Sec was observed along with a tenfold increase in selenoprotein yield compared to Sec insertion at the three stop codons. This high-fidelity sense-codon recoding mechanism was demonstrated for Escherichia coli formate dehydrogenase and recombinant human thioredoxin reductase and confirmed by independent biochemical and biophysical methods. Although Sec insertion at UGA is known to compete against protein termination, it is surprising that the Sec machinery has the ability to outcompete abundant aminoacyl-tRNAs in decoding sense codons. The findings have implications for the process of translation and the information storage capacity of the biological cell.

Figure 1

Recoding the genetic code with Sec. The canonical genetic code table is overlaid on a single agar plate spotted with 64 E. coli FDH_H variants. In each case, an E. coliΔselAΔselBΔfdhF deletion parent strain was complemented with E. coli selA, selB, and each of the 64 fdhF ₁₄₀NNN codon mutants and tRNA^Sec_NNN variants with the respective cognate anticodon. The capacity of each strain to recode the indicated codon to Sec is evidenced by an in vivo BV reduction assay in which purple colored cells express active Sec-containing FDH_H.

Figure 2

Quantitation of codon recoding with Sec. A) The relative yield of the 64 FDH_{H 140}NNN variants (wild type FDH_{H 140}UGA = 1) is plotted versus specific activity (relative to wild type FDH_{H 140}UGA = 100%). Activity, yield values and standard deviations are shown in Table S1. Different levels of specific activity observed for the 64 FDH_H variants are the result of the partial incorporation of the respective canonical AAs (see Table S2). B) MS/MS identification of UAC-encoded Sec. The spectrum of the Sec-containing tryptic peptide, peptide sequence, and position of the observed b- and y-ions are indicated (see also Table S2). Sec (U) was identified by the Mascot protein identification software as a Se-IAN-Cys modification of Cys; selenium alkylation (Se-CH₂-CONH₂).

Figure 2

Quantitation of codon recoding with Sec. A) The relative yield of the 64 FDH_{H 140}NNN variants (wild type FDH_{H 140}UGA = 1) is plotted versus specific activity (relative to wild type FDH_{H 140}UGA = 100%). Activity, yield values and standard deviations are shown in Table S1. Different levels of specific activity observed for the 64 FDH_H variants are the result of the partial incorporation of the respective canonical AAs (see Table S2). B) MS/MS identification of UAC-encoded Sec. The spectrum of the Sec-containing tryptic peptide, peptide sequence, and position of the observed b- and y-ions are indicated (see also Table S2). Sec (U) was identified by the Mascot protein identification software as a Se-IAN-Cys modification of Cys; selenium alkylation (Se-CH₂-CONH₂).

Figure 3

Recoding the Tyr codon UAC to Sec in human TrxR1. A) Schematic representation of the expression construct for human TXNRD1. An E. coli fdhF SECIS element is attached directly after the UAA codon of TXNRD1 in the 3′ untranslated region (UTR). B) In vitro activity assay of recombinant human TrxR ₅₅₀UGA and TrxR ₅₅₀UAC co-expressed with tRNA^Sec_UCA and tRNA^Sec_GUA, respectively. Error bars show standard deviations of quadruplicate experiments. C) MS/MS identification of Sec incorporation into TrxR ₅₅₀UAC. The spectrum of the Sec-containing peptide, the peptide sequence and the positions of the observed b- and y-ions are indicated. Sec was identified by MASCOT as a Se-IAN-Cys modification of Cys; selenium alkylation (Se-CH₂-CONH₂).

Scheme 1

Codon recoding with Sec. A) Synthesis of the E. coli Sec-containing formate dehydrogenase (FDH) from the wild type fdhF gene requires recoding the UGA stop codon at position 140 to Sec by Sec-tRNA^Sec, elongation factor SelB, and the SECIS mRNA hairpin. Replacing the UGA with any of the 64 NNN triplets at codon 140 in fdhF and co-expressing a cognate Sec-tRNA^Sec_NNN is expected to yield SelB and SECIS-dependent sense codon-recoded Sec-containing FDH_H (B) that may compete with canonical AA insertion (C) directed by native aminoacyl-tRNAs and EF-Tu dependent protein synthesis. In this case (C), SECIS is still present in the fdhF mRNA, but SECIS is not expected to interact with EF-Tu directed decoding.