UGA Codon Position Affects the Efficiency of Selenocysteine Incorporation into Glutathione Peroxidase-1

Wu Wen,Sherri L Weiss,Roger A Sunde

doi:10.1074/jbc.273.43.28533

Abstract

A UGA codon and a selenocysteine insertion sequence in the 3'-untranslated region are the only established mRNA elements necessary for selenocysteine (Sec or U) incorporation during translation. These two elements, however, do not universally confer efficient Sec incorporation. The objective of this study was to systematically examine the effect of UGA codon position on efficiency of Sec insertion. In a glutathione peroxidase-1 (F-GPX1) expression vector, the UGA at the native position (U47) was mutated to a cysteine codon, and codons for Ser-7, Ser-12, Ser-18, Ser-29, Ser-45, Ser-93, Cys-154, Val-172, Ser-178, and Ser-195 were individually mutated to UGA and transiently expressed in COS-7 cells. 75Se incorporation at the 11 positions was 31, 72, 54, 105, 90, 100, 146, 135, 13, 11, and 43%, respectively, of 75Se incorporation at U47, suggesting that Sec is more efficiently incorporated at UGA codons positioned in the middle of the coding region rather than close to the 5' or 3' ends. Ribonuclease protection showed that these differences were not due to differences in mRNA level. When the green fluorescence protein (GFP) coding region was placed in-frame at the 5' or 3' ends of the coding region in F-GPX1 to produce chimeric 50-51-kDa GFP/GPX1 proteins, Sec incorporation at UGA codons, formerly close to the 5' or 3' ends, was increased to levels comparable to the UGA at U47. Insertion of GFP after the UAA-stop was just as effective in increasing Sec insertion efficiency as GFP inserted before the stop. These studies used a recombinant expression model that incorporated Sec at non-native UGA codons at rates equal to those of endogenous glutathione peroxidase-1 and showed that the efficiency of Sec incorporation can be modulated by UGA position; Sec incorporation at high efficiency appears to require that the UGA be >21 nucleotides from the AUG-start and >204 nucleotides from the selenocysteine insertion sequence element.

Highlights

Containing enzyme [1], and this discovery provided a biochemical role for selenium
Cells transfected with gpx1 without FLAG epitope (GPX1) did not significantly alter 75Se incorporation into thioredoxin reductase (TR) or glutathione peroxidase-4 (GPX4) when compared with vector-transfected cells, but there was a marked increase in 75Se incorporation into GPX1
GPX1 activity was increased 2-fold over wild-type cells; similar modest increases in selenoenzyme activity are reported for GPX1-transfected cells that possess endogenous GPX1 activity (39 – 42). 75Se incorporation into GPX1, expressed relative to ␤-galactosidase activity to normalize for transfection efficiency, was 3-fold (61 versus 21%) higher than the vector-alone transfectants, showing that this basal construct directed significant 75Se incorporation into GPX1 in COS-7 cells

Summary

Introduction

Containing enzyme [1], and this discovery provided a biochemical role for selenium. The selenium is present as selenocysteine (Sec or U) incorporated into the peptide backbone, and Sec is located at the active site of GPX1 [2, 3]. The carbon skeleton of the Sec is provided by serine [29]; serine, esterified to a unique tRNAUSeCrA3Sec in both eukaryotes and prokaryotes, and selenophosphate, synthesized by selenophosphate synthetase, are the substrates used to form Sec-tRNAUSeCrA3Sec [24, 30] This reaction is catalyzed by selenocysteine synthetase (SELA) in bacteria [31, 32], but the enzyme is yet to be characterized in eukaryotes. One study, which focused directly on the base following the UGA, reported that Sec incorporation could change as much as 3-fold depending on the identity of the fourth base These reports suggest that additional cis-elements may be required for efficient Sec translation or that the assembly of the required components at the UGA in combination with competition with release factors may be inefficient relative to the incorporation of other amino acids

Objectives

Results

Conclusion