Selenocysteine (the 21st amino acid) is present in all three kingdoms of life. In bacteria, its incorporation into proteins requires highly specific machinery including elongation factor SelB (Forchhammer et al.
). SelB is able to interact with the ribosome, selenocysteyl-tRNASec
and guanine nucleotides via
its N-terminal part, which is homologous to the three domains of elongation factor Tu (EF-Tu). An extra C-terminal domain (SelB-C) binds an mRNA hairpin called SECIS (selenocysteine-insertion sequence), which serves as a signal to deliver Sec-tRNASec
to a UGA codon at the ribosomal A site. Hence, a UGA codon with a downstream SECIS is recoded as a Sec codon instead of being used as a termination signal. The crystal structure of SelB-C from Moorella thermoacetica
(Selmer & Su, 2002
) and the solution structure of the Escherichia coli
SECIS hairpin (Fourmy et al.
) have been reported. SelB-C is composed of four consecutive winged-helix motifs (WH motifs), a module that is found in many DNA-binding proteins (Gajiwala & Burley, 2000
). The crystal structure of the M. thermoacetica
SelB WH3/4 C-terminal domain in complex with SECIS (Yoshizawa et al.
) has shed light on the molecular basis of this interaction. The WH4 domain recognizes the extruded nucleotides G23 and U24 at the tip of the SECIS stem loop as well as five consecutive helical phosphates on the 5′ side of the hairpin loop. However, the affinity between M. thermoacetica
SelB and SECIS is in the micromolar range (Yoshizawa et al.
), which is three orders of magnitude lower than in the case of E. coli
SelB (nanomolar range; Thanbichler et al.
). This observation raises the possibility that the high affinity of E. coli
SelB for mRNA originates from the use of the modular organization of the WH motifs. This is supported by the identification of a bulged uracil residue (U17) located outside the main protein-binding site as being crucial for high-affinity binding of mRNA in E. coli
SECIS (Hüttenhofer et al.
; Liu et al.
). In the previous crystal structure of a WH3/4–SECIS RNA complex from M. thermoacetica
, the predicted position of the bulged uracil lies in the vicinity of the WH3 domain (Yoshizawa et al.
). Thus, the WH3 module in E. coli
SelB may directly establish specific contacts with SECIS RNA.
In this paper, we report the production and crystallization of the minimum fragment of the RNA-binding domain (SelB-WH3/4; residues 478–614) of SelB from E. coli in complex with the SECIS mRNA hairpin. This will allow the investigation of the RNA-binding activity of the two consecutive WH motifs (WH3-WH4) in E. coli SelB, for which abundant genetic, biochemical and kinetic data are available.