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

Abstract

Selenocysteine (Sec) incorporation requires the TGA opal codon and a downstream Sec insertion sequence (SECIS), which can be partially randomized and cloned into M13 pIII fusion constructs for phage display. This combinatorial approach provides a convenient non-radioactive assay that couples phage production to opal suppression. Two SECIS libraries were prepared, with the immediate downstream nucleotide either randomized (TGAN) or fixed as thymidine (TGAT). The TGAN library resulted in a majority of clones with a downstream 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, it did increase the level of Sec insertion, as shown by the chemical reactivity of the resulting phage. The TGAT phage library yielded clones with strictly selenium-dependent phage production and reactivity consistent with the presence of Sec. These clones were prone to spontaneous mutation upon further propagation, however, resulting in loss of the selenium-dependent phenotype. We conclude that the immediate downstream nucleotide determines whether the endogenous opal suppression pathway competes with co-translational Sec insertion.

Figure 1

Biosynthetic pathway for co-translational selenocysteine incorporation. SelC, the opal codon-specific Sec tRNA, is first charged with serine. ATP-dependent SelD catalysis transforms environmental selenite to an activated Se-phosphate species. This species is utilized by SelA to displace the serine hydroxyl with a selenol moiety, forming a Sec-charged tRNA that recognizes the UGA opal codon. In the presence of GTP, the SelB elongation factor effects Sec translation by binding both the Sec-tRNA and the mRNA SECIS.

Figure 2

The E.coli formate dehydrogenase SECIS, with permissible mutations as reported in Heider et al. (5), Liu et al. (7) and Klug et al. (12).

Figure 3

The randomized library inserts expressed as pIII fusions in this report. N = A, G, C or U; K = G or U.

Figure 4

Plating results showing Se-dependent and Se-independent growth of phage clones TGAT-13, TGAT-10 and TGAN-8 (sequences in Tables 1 and 2). Left column, no supplemental Se in plating medium; right column, 2 µM sodium selenite added to plating medium.

Figure 5

Immunoblots showing specific chemical modification of phage displaying selenopeptides. Individual library clones from the TGAN library (A) or TGAT library (B) were amplified with or without 2 µM supplemental sodium selenite as indicated. Phage were modified as described in Materials and Methods. *Amplification of clone TGAT-1 in unsupplemented medium resulted in a TGA→TGG point mutation.

Figure 6

Opal suppression flow chart, based on experimental data derived from expressing SECIS variants as N-terminal fusions of M13 pIII.