Dynamics and efficiency invivo of UGA-directed selenocysteine insertion at the ribosome

Abstract

The kinetics and efficiency of decoding of the UGA of a bacterial selenoprotein mRNA with selenocysteine has been studied in vivo. A gst-lacZ fusion, with the fdhF SECIS element ligated between the two fusion partners, gave an efficiency of read-through of 4-5%; overproduction of the selenocysteine insertion machinery increased it to 7-10%. This low efficiency is caused by termination at the UGA and not by translational barriers at the SECIS. When the selenocysteine UGA codon was replaced by UCA, and tRNASec with anticodon UGA was allowed to compete with seryl-tRNASer1 for this codon, selenocysteine was found in 7% of the protein produced. When a non-cognate SelB-tRNASec complex competed with EF-Tu for a sense codon, no effects were seen, whereas a non-cognate SelB-tRNASec competing with EF-Tu-mediated Su7-tRNA nonsense suppression of UGA interfered strongly with suppression. The induction kinetics of beta-galactosidase synthesis from fdhF'-'lacZ gene fusions in the absence or presence of SelB and/or the SECIS element, showed that there was a translational pause in the fusion containing the SECIS when SelB was present. The results show that decoding of UGA is an inefficient process and that using the third dimension of the mRNA to accommodate an additional amino acid is accompanied by considerable quantitative and kinetic costs.

Efficiency of Mammalian Selenocysteine Incorporation

“…This is reported to be the case for bacterial Sec incorporation where maximum efficiency is reported to be 7-10% (13). In mammals, several selenoproteins are made at very high levels, particularly PHGPx in the mammalian testis (14), suggesting that efficiency may be much higher than that found in prokaryotes.…”

Regulation of Selenocysteine Incorporation into the Selenium Transport Protein, Selenoprotein P

“…To observe the predominant termination products that are the result of the inherently inefficient Sec incorporation in RRL, we first translated each of these mutant mRNAs in the presence of [ 35 S]Met (Fig. 2B, lanes [1][2][3][4][5][6][7][8][9][10]. This allowed detection of truncation products corresponding to termination at Sec codons 2 to 10 ( Fig.…”

“…In addition to the SECIS, three other factors are required: SECIS binding protein 2 (SBP2), a dedicated eukaryotic elongation factor (eEFSec), and the selenocysteyl tRNA Sec (SectRNA Sec ) (4 -7). In bacteria, the maximum Sec incorporation efficiency has been reported to be 7-10% (8). The efficiency of Sec incorporation in the rabbit reticulocyte lysate in vitro translation system was also reported to be ϳ10% (9), but the frequency of early termination at the Sec codon for endogenous selenoproteins in mammals remains unknown.…”

Endless possibilities: translation termination and stop codon recognition

“…Improving this context reduces selenocysteine incorporation at the expense of termination (McCaughan et al, 1995). However, readthough efficiencies of 4-5 % at the E. coli fdhF UGA selenocysteine codon and 75 % at the mammalian deiodinase internal UGA codon indicate that recoding has variable efficiency (Suppmann et al, 1999 ;McCaughan et al, 1995). Apart from nucleotide context, the structured SECIS elements in both eukaryote and prokaryote systems are essential for UGA recoding.…”

“…Apart from nucleotide context, the structured SECIS elements in both eukaryote and prokaryote systems are essential for UGA recoding. In prokaryotes, SECIS elements do not act simply to increase the local concentration of tRNA Sec -SelB complex at the UGA codon, thus outcompeting RF2 ; rather the SECIS element is required for delivery of the tRNA Sec -SelB-GTP ternary complex to the ribosome, and overexpressing the SelB-tRNA combination does not increase the efficiency of incorporation (Suppmann et al, 1999). This delivery mechanism, together with context effects, seems to be sufficient to much reduce alternative termination reactions.…”