Facile Recoding of Selenocysteine in Nature

Abstract

Selenocysteine (Sec or U) is encoded by UGA, a stop codon reassigned by a Sec-specific elongation factor and a distinctive RNA structure. To discover possible code variations in extant organisms we analyzed 6.4 trillion base pairs of metagenomic sequences and 24 903 microbial genomes for tRNA(Sec) species. As expected, UGA is the predominant Sec codon in use. We also found tRNA(Sec) species that recognize the stop codons UAG and UAA, and ten sense codons. Selenoprotein synthesis programmed by UAG in Geodermatophilus and Blastococcus, and by the Cys codon UGU in Aeromonas salmonicida was confirmed by metabolic labeling with (75) Se or mass spectrometry. Other tRNA(Sec) species with different anticodons enabled E. coli to synthesize active formate dehydrogenase H, a selenoenzyme. This illustrates the ease by which the genetic code may evolve new coding schemes, possibly aiding organisms to adapt to changing environments, and show the genetic code is much more flexible than previously thought.

Keywords: genetic code; metagenome; selenocysteine; sense codon recoding; synthetic biology.

Figure 1. Non-canonical selenocysteine assignments in nature

(A) The tRNA^Sec search pipeline and the manually curated output. The non-canonical tRNA^Sec sequences are grouped by codon recognition; their numbers are given with their (putative) bacterial origins from phylogenetic inference. Green color indicates results from whole genomes, while blue represents results from only metagenomic data. For comparison the number of canonical tRNA^Sec_UCA sequences is shown (most of them were not curated). “Y” and “R” denote “C or U” and “A or G”, respectively. (B) Inferred cloverleaf structures of non-canonical tRNA^Sec species. The nucleotide polymorphism among the same tRNA^Sec group is indicated with green letters. The Sec codons and the SECIS elements of formate dehydrogenase mRNAs are shown.

Figure 2. Recoding of UAG and cysteine codons to selenocysteine

(A) Metabolic ⁷⁵Se labeling of G. obscurus and B. saxobsidens cells. Crude extracts were resolved by SDS-PAGE, and their putative selenoproteins were visualized by PhosphorImager analysis. The results of peptide mass fingerprinting (PMF) analyses of the proteins in the excised gel bands are shown to the right of the bands. (B) FDH_H expression in E. coli ΔselABC ΔfdhF cells with the G. obscurus selABC genes and a chimeric fdhF(140TAG) gene variant having a G. obscurus SECIS element with a few nucleotide modifications shown in red. The selC(CUA) genes express tRNA^Sec_CUA. The expressed selenoprotein FDH_H reduced benzyl viologen, resulting in a purple color. (C) FDH_H activity of A. salmonicida subsp. pectinolytica 34mel cells. (D) Metabolic ⁷⁵Se labeling of the FDH_H of 34mel. (E) The procedure of sample preparation for the LC-MS/MS analysis of the FDH_H selenoprotein. (F) PMF confirms Sec incorporation at codon 140 in the recombinant FDH_H. (G) tRNA^Sec_GCA gene (selC) locus in a metagenomic contig. (H) In vivo FDH_H assays in E. coli ΔselABC ΔfdhF cells with the selABC genes of the metagenomic contig and a chimeric fdhF(140TGC) gene carrying the contig’s SECIS element with a few nucleotide modifications shown in red. The two transformed strains boxed were metabolically labeled with ⁷⁵Se, and the radioactive FDH_H proteins were analyzed by SDS-PAGE and autoradiography or western blotting (WB).