Journal Article

Interaction of the Escherichia Coli fdhF mRNA Hairpin Promoting Selenocysteine Incorporation with the Ribosome

Alexander Hüttenhofer, Johann Heider and August Böck

in Nucleic Acids Research

Volume 24, issue 20, pages 3903-3910
Published in print October 1996 | ISSN: 0305-1048
Published online October 1996 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/24.20.3903
Interaction of the Escherichia Coli fdhF mRNA Hairpin Promoting Selenocysteine Incorporation with the Ribosome

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The codon UGA located 5′ adjacent to an mRNA hairpin within fdhF mRNA promotes the incorporation of the amino acid selenocysteine into formate dehydrogenase H of Escherichia coli. The loop region of this mRNA hairpin has been shown to bind to the special elongation factor SELB, which also forms a complex with selenocysteinyl-tRNASec and GTP. We designed seven different mRNA constructs derived from the fdhF mRNA which contain a translation initiation region including an AUG initiation codon followed by no, one, two, three, four, five or six UUC phenylalanine codon(s) and the UGA selenocysteine codon 5′ adjacent to the fdhF mRNA hairpin. By binding these different mRNA constructs to 30S ribosomal subunits in vitro we attempted to mimic intermediate steps of elongation of a structured mRNA approaching the ribosome by one codon at a time. Toeprint analysis of the mRNA-ribo-some complexes showed that the presence of the fdhF mRNA hairpin strongly interferes with binding of the fdhF mRNA to 30S ribosomal subunits as soon as the hairpin is placed closer than 16 bases to the ribosomal P-site. Binding is reduced up to 25-fold compared with mRNA constructs where the hairpin is located outside the ribosomal mRNA track. Surprisingly, no toeprint signals were observed in any of our mRNA constructs when tRNASec was used instead of tRNAfMet. Lack of binding of selenocysteinyl-tRNASec to the UGA codon was attributed to steric hindrance by the fdhF mRNA hairpin. By chemical probing of the shortest mRNA construct (AUG-UGA-fdhF hairpin) bound to 30S ribosomal subunits we demonstrate that the hairpin structure is not unfolded in the presence of ribosomes in vitro; also, this mRNA is not translated in vivowhen fused in-frame 5′ of the lacZgene. Therefore, our data indicate that the fdhFmRNA hairpin has to be unfolded during elongation prior to entering the ribosomal mRNA track and we propose that the SELB binding domain within the fdhF mRNA is located outside the ribosomal mRNA track during decoding of the UGA selenocysteine codon by the SELB-selenocysteinyl-tRNASec-GTP complex.

Journal Article.  4535 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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