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.

FIG. 1

Minimal E. coli fdhF SECIS required for SelB binding and selenocysteine incorporation. The upper stem-loop structure (boxed) is the minimal region required for SelB binding (13). UGA-directed selenocysteine incorporation requires that this structure be located 11 nucleotides (nt) from the UGA codon (bold) (18). The U residue at position 17 is bulged (12, 18). The pairing of boxes C₂₀ and G₂₇ is questionable (18) and is therefore designated by a dot instead of by a dash.

FIG. 2

Location of the mutations in the upper stem and loop of the E. coli fdhF SECIS on the plasmids used in this study. The mutated nucleotides are boxed. In pZL44, U₁₈ is bulged (circle). The numbers represent the distance of the nucleotide from the UGA codon of E. coli fdhF SECIS.

FIG. 3

Schematic representation of the region in SelB in which the mutations are clustered that suppress the mutated U₁₇ bulged nucleotide in E. coli fdhF SECIS. selB DNA corresponding to SelB domains 4a (amino acids [aa] 343 to 474) and 4b (amino acids 472 to 614) (13) (dotted region) was subjected to PCR random mutagenesis. Selection was made for mutations in selB that could suppress mutations in the SECIS U₁₇ bulged nucleotide; these mutations were subsequently sequenced (Table 2 and Materials and Methods). The selected single mutations are located in 28 amino acids of the 4b region (between 556 and 583 of SelB) (shaded rectangle). Mutated amino acids are underlined.

FIG. 4

Selected mutations in selB suppress mutations in different positions of the upper stem-loop of E. coli SECIS. E. coli WL83100 cells were double transformed, first by each of plasmids pL24A, pZL44, pZL38, pZL42, and pZL70 and then by each of plasmids p70bm[1], p70bm[2], p70bm[3], p70bm[6], p70bm[7], p70bm[9], p42bm[4], or the wild-type pLC1. The level of suppression (represented by the number above the columns) was determined by the level of β-galactosidase as described in Table 2, footnote e.