Interplay between termination and translation machinery in eukaryotic selenoprotein synthesis

Termination of translation in eukaryotes is catalyzed by eRF1, the stop cod on recognition factor, and eRF3, an eRF1 and ribosome-dependent GTPase. In selenoprotein mRNAs, UGA codons, which typically specify termination, serve an alternate function as sense codons. Selenocysteine incorporation involv es a unique tRNA with an anticodon complementary to UGA, a unique elongatio n factor specific for this tRNA, and cis-acting secondary structures in sel enoprotein mRNAs, termed SECIS elements. To gain insight into the interplay between the selenocysteine insertion and termination machinery, we investi gated the effects of overexpressing eRF1 and eRF3, and of altering UGA codo n context, on the efficiency of selenoprotein synthesis in a transient tran sfection system. Overexpression of eRF1 does not increase termination at na turally occurring selenocysteine codons. Surprisingly, selenocysteine incor poration is enhanced. Overexpression of eRF3 did not affect incorporation e fficiency. Coexpression of both factors reproduced the effects with eRF1 al one. Finally, we show that the nucleotide context immediately upstream and downstream of the UGA codon significantly affects termination to incorporat ion ratios and the response to eRF overexpression. Implications for the mec hanisms of selenocysteine incorporation and termination are discussed. (C) 2001 Academic Press.