The Bulged Nucleotide in the Escherichia coli Minimal Selenocysteine Insertion Sequence Participates in Interaction with SelB: a Genetic Approach

Abstract

The UGA codon, which usually acts as a stop codon, can also direct the incorporation into a protein of the amino acid selenocysteine. This UGA decoding process requires a cis-acting mRNA element called the selenocysteine insertion sequence (SECIS), which can form a stem-loop structure. In Escherichia coli, selenocysteine incorporation requires only the 17-nucleotide-long upper stem-loop structure of the fdhF SECIS. This structure carries a bulged nucleotide U at position 17. Here we asked whether the single bulged nucleotide located in the upper stem-loop structure of the E. coli fdhF SECIS is involved in the in vivo interaction with SelB. We used a genetic approach, generating and characterizing selB mutations that suppress mutations of the bulged nucleotide in the SECIS. All the selB suppressor mutations isolated were clustered in a region corresponding to 28 amino acids in the SelB C-terminal subdomain 4b. These selB suppressor mutations were also found to suppress mutations in either the loop or the upper stem of the E. coli SECIS. Thus, the E. coli SECIS upper stem-loop structure can be considered a "single suppressible unit," suggesting that there is some flexibility to the nature of the interaction between this element and SelB.The UGA codon, which usually acts as a stop codon, can also direct incorporation of the amino acid selenocysteine (for reviews, see references 4, 5, 7, and 25). This UGA decoding process requires a cis-acting mRNA element called the selenocysteine insertion sequence (SECIS), which can form a stemloop structure (4, 9, 15; for reviews, see references 2 and 20).In Escherichia coli, a number of genes have been identified in which the UGA directs the incorporation of selenocysteine. These include genes fdhF (27) and fdnG (3), encoding the selenocysteine-containing enzymes formate dehydrogenase H and N, respectively. Immediately downstream from the selenocysteine-specifying UGA in the mRNA of each of these polypeptides is found a SECIS that has been described as consisting of at least 40 nucleotides capable of forming a stem-loop RNA structure (2, 9). In later work, it was suggested that an extended fdhF SECIS was required, consisting of an additional helix of 7 bp in which the U and G residues of the UGA codon are included and the A residue is bulged out (10). After carrying out an extensive mutational analysis of the fdhF SECIS DNA, we found that for in vivo UGA-directed selenocysteine incorporation, there is no requirement for the whole stem-loop RNA structure of the E. coli fdhF SECIS (including the extended form) (18), as thought previously. Instead, the 17-bp upper stem-loop structure is sufficient to permit selenocysteine incorporation on the condition that it is located 11 nucleotides downstream from the UGA codon (Fig. 1). This mini upper stem-loop structure contains a bulged nucleotide, a U residue, located 17 nucleotides downstream from the UGA (Fig. 1). Selenocysteine incorporation into an fdhF-lacZЈ fusion polypeptide depends on both (i) the specificity of nucleotide 17 as a...

Structural Insight into a Molecular Switch in Tandem Winged-helix Motifs from Elongation Factor SelB

“…20,43 The E. coli WH3/4 structure described here allows us to map the mutations directly on the structure (Supplementary Data, Figure S6). Most mutations that suppress substitutions of the bulged nucleotide U17 in SECIS are clustered in a region corresponding to 28 amino acid residues in the WH4 domain.…”

The three-dimensional structure of the Moorella thermoacetica selenocysteine insertion sequence RNA hairpin and its interaction with the elongation factor SelB

“…In E. coli a number of SECIS molecules have been identified, and their function has been the subject of extensive biochemical analysis (Huttenhofer et al 1996;Klug et al 1999;Li et al 2000;Thanbichler et al 2000). The best-studied SECIS RNA molecules are from the genes encoding the proteins formate dehydrogenase H and N, fdhF and fdnG, respectively.…”

Identification and characterization of a selenoprotein family containing a diselenide bond in a redox motif

“…A minimal SECIS region required for binding E. coli SelB is a 17-nt stem-loop separated from Sec UGA by 11 nt. The 17-nt stem-loop also contains a bulged U in the stem at a distance Ϸ17 nt from the UGA codon and a GU dinucleotide in the apical loop at a distance Ϸ23-24 nt from the UGA codon (25)(26)(27). Natural bacterial SelL SECIS elements did not have the bulged U; the distances between the first UGA codon and the exposed GU were 30-31 nt and the distances between the second UGA and the GU were 20-21 nt (Fig.…”

“…In an attempt to increase expression of Sec-containing proteins in E. coli (46), two different SECIS structures with bulged nucleotides were produced by site-directed mutagenesis to yield pGEX-SelL-SECISdel and pGEX-SelL-SECISins constructs ( Fig. 1B; and see SI Table 3 for primer sequences).…”