Recoding of the selenocysteine UGA codon by cysteine in the presence of a non-canonical tRNA^Cysand elongation factor SelB

Abstract

In many organisms, the UGA stop codon is recoded to insert selenocysteine (Sec) into proteins. Sec incorporation in bacteria is directed by an mRNA element, known as the Sec-insertion sequence (SECIS), located downstream of the Sec codon. Unlike other aminoacyl-tRNAs, Sec-tRNA is delivered to the ribosome by a dedicated elongation factor, SelB. We recently identified a series of tRNA-like tRNA genes distributed across Bacteria that also encode a canonical tRNA. These tRNAs contain sequence elements generally recognized by cysteinyl-tRNA synthetase (CysRS). While some of these tRNAs contain a UCA Sec anticodon, most have a GCA Cys anticodon. tRNA with GCA anticodons are known to recode UGA codons. Here we investigate the clostridial Desulfotomaculum nigrificans tRNA-like tRNA, and show that this tRNA is acylated by CysRS, recognized by SelB, and capable of UGA recoding with Cys in Escherichia coli. We named this non-canonical group of tRNA as 'tRNA' (Recoding with Cys). We performed a comprehensive survey of tRNA genes to establish their phylogenetic distribution, and found that, in a particular lineage of clostridial Pelotomaculum, the Cys identity elements of tRNA had mutated. This novel tRNA, which contains a UCA anticodon, is capable of Sec incorporation in E. coli, albeit with lower efficiency relative to Pelotomaculum tRNA. We renamed this unusual tRNA derived from tRNA as 'tRNA' (Recoding with Sec). Together, our results suggest that tRNA and tRNA may serve as safeguards in the production of selenoproteins and - to our knowledge - they provide the first example of programmed codon-anticodon mispairing in bacteria.

Bioinformatic Prediction of an tRNASec Gene Nested inside an Elongation Factor SelB Gene in Alphaproteobacteria

“…The two ORFs associating with the overlapping selBC gene were named ORF1 and ORF2 in this study. As reported previously [12], Methylophila spp. use a tRNA Sec with a non-canonical discriminator base A73 instead of G73.…”

“…It is known that a megaplasmid pSymA of Sinorhizobium ( Rhizobium ) meliloti encode a fdoGHI (for FDH-O) and selABCD gene cluster [ 10 , 11 ]. In our previous study on non-canonical SelB sequences [ 12 ], it was found that Rhizobiales SelB sequences in the public databases are annotated to have a C-terminal extension compared to other bacterial SelB sequences ( Figure 1 A) [ 13 ]. In the present study, a careful analysis of these Rhizobiales SelB sequences was performed to reveal the possible reason.…”

A cysteinyl-tRNA synthetase variant confers resistance against selenite toxicity and decreases selenocysteine misincorporation

“…E. coli WT CysRS was purified as described previously (47). Site-directed mutagenesis was used to replace His with Asn at position 235 of E. coli CysRS.…”

“…Plates were run in buffer containing 0.75 KH 2 PO 4 and 1 M urea for ϳ45 min. Sec (50 mM) was freshly prepared as described previously (49,50) tRNA aminoacylation assays E. coli tRNA Cys was transcribed in vitro using T7 RNA polymerase and labeled at the 3Ј-end adenosine with [ 32 P]phosphate as described previously (47,51). Aminoacylation experiments were carried out in buffer containing 50 mM HEPES (pH 7.3), 10 mM ATP, 2 mM DTT, 10 mM MgCl 2 , 50 mM KCl, 20 M E. coli tRNA Cys (containing trace amounts of 32 P-labeled tRNA), and varying concentrations of Sec and Cys (3-600 M).…”

Transfer RNA function and evolution

“…Fu et al reviewed the underlying molecular biology of translation with selenocysteine and they address challenges associated with engineering designer selenoproteins [7]. In a related study, Vargas-Rodriguez et al uncovered an unusual or non-canonical tRNA Cys that co-opts the selenocysteine machinery to incorporate cysteine instead [8]. The authors characterized a novel tRNA Sec -like tRNA Cys from Desulfotomaculum nigrificans that recodes UGA codons with cysteine instead of selenocysteine and dubbed this new molecule tRNA ReC to indicate its ability to recode with cysteine.…”