The efficiency of Escherichia coli selenocysteine insertion is influenced by the immediate downstream nucleotide

Selenocysteine (Sec) incorporation requires the TGA opal codon and a downst ream Sec insertion sequence (SECIS), which can be partially randomized and cloned into M13 pill fusion constructs for phage display, This combinatoria l approach provides a convenient non-radioactive assay that couples phage p roduction to opal suppression. Two SECIS libraries were prepared, with the immediate downstream nucleotide either randomized (TGAN) or fixed as thymid ine (TGAT), The TGAN library resulted in a majority of clones with a downst ream purine and selenium-independent phage production, implicating the endo genous tryptophan-inserting opal suppression pathway. Although the addition of sodium selenite to the growth medium did not affect phage production, i t did increase the level of Sec insertion, as shown by the chemical reactiv ity of the resulting phage, The TGAT phage library yielded clones with stri ctly selenium-dependent phage production and reactivity consistent with the presence of Sec, These clones were prone to spontaneous mutation upon furt her propagation, however, resulting in loss of the selenium-dependent pheno type, We conclude that the immediate downstream nucleotide determines wheth er the endogenous opal suppression pathway competes with co-translational S ec insertion.