Nucleic Acids Research, Vol 24, Issue 20 3903-3910, Copyright © 1996 by Oxford University Press
A Huttenhofer, J Heider and A Bock
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-tRNA(Sec) 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-ribosome 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 tRNA(Sec) was used instead of tRNA(fMet). Lack of binding
of selenocysteinyl-tRNA(Sec) 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 vivo when fused
in-frame 5' of the lacZ gene. Therefore, our data indicate that the fdhF
mRNA 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-tRNA(Sec)-GTP
complex.
ARTICLES
Interaction of the Escherichia coli fdhF mRNA hairpin promoting selenocysteine incorporation with the ribosome
Lehrstuhl fur Mikrobiologie der Universitat Munchen, Germany.
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