AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo.

Institute of Physical and Chemical Biology, Lutetia Parisorum, Île-de-France, France
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 07/1987; 84(12):4022-5. DOI: 10.1073/pnas.84.12.4022
Source: PubMed

ABSTRACT We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo.


Available from: Mathias Springer, Jun 13, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Expression of the gene pcnB, encoding the dispensable Escherichia coli poly(A) polymerase (PAPI), which is toxic when overproduced, was investigated. Its promoter was identified and found to be moderately strong when used to express a beta-galactosidase reporter. Expression levels were not affected by increasing or decreasing PcnB concentration. Translation of pcnB was found to initiate from the non-canonical initiation codon AUU. The only other coli gene reported to use AUU as initiation codon is infC, which encodes the initiation factor IF-3. AUU, in common with other rarely used initiation codons, is discriminated against by IF-3, resulting in the aborting of most AUU-promoted initiation events. This enables AUU to form part of an autoregulatory circuit controlling IF-3 production. We show that InfC discrimination reduces PcnB production fivefold. This is the first instance of this mechanism being used to limit severely the production of a potentially toxic product.
    Molecular Microbiology 07/2002; 44(5):1287-98. DOI:10.1046/j.1365-2958.2002.02945.x · 5.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In this study, we have examined the influence of initiation factors on translation initiation of leaderless mRNAs whose 5'-terminal residues are the A of the AUG initiating codon. A 1:1 ratio of initiation factors to ribosomes abolished ternary complex formation at the authentic start codon of different leaderless mRNAs. Supporting this observation, in vitro translation assays using limiting ribosome concentrations with competing leaderless lambda cl and Escherichia coli ompA mRNAs, the latter containing a canonical ribosome binding site, revealed reduced cl synthesis relative to OmpA in the presence of added initiation factors. Using in vitro toeprinting and in vitro translation assays, we show that this effect can be attributed to IF3. Moreover, in vivo studies revealed that the translational efficiency of a leaderless reporter gene is decreased with increased IF3 levels. These studies are corroborated by the observed increased translational efficiency of a leaderless reporter construct in an infC mutant strain unable to discriminate against non-standard start codons. These results suggest that, in the absence of a leader or a Shine-Dalgarno sequence, the function(s) of IF3 limits stable 30S ternary complex formation.
    Molecular Microbiology 02/1999; 31(1):67-77. DOI:10.1046/j.1365-2958.1999.01147.x · 5.03 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Joining of the large, 50S, ribosomal subunit to the small, 30S, ribosomal subunit initiation complex (30S IC) during bacterial translation initiation is catalyzed by initiation factor (IF) 2. Because the rate of subunit joining is coupled to the IF-, transfer RNA (tRNA)-, and messenger RNA (mRNA) codon composition of the 30S IC, the subunit joining reaction functions as a kinetic checkpoint that regulates the fidelity of translation initiation. Recent structural studies suggest that the conformational dynamics of the IF2•tRNA sub-complex that forms on the intersubunit surface of the 30S IC may play a significant role in the mechanisms that couple the rate of subunit joining to the IF-, tRNA-, and codon composition of the 30S IC. To test this hypothesis, we have developed a single-molecule fluorescence resonance energy transfer signal between IF2 and tRNA that has enabled us to monitor the conformational dynamics of the IF2•tRNA sub-complex across a series of 30S ICs. Our results demonstrate that 30S ICs that undergo rapid subunit joining display a high affinity for IF2 and an IF2•tRNA sub-complex that primarily samples a single conformation. In contrast, 30S ICs that undergo slower subunit joining exhibit a decreased affinity for IF2 and/or a change in the conformational dynamics of the IF2•tRNA sub-complex. These results strongly suggest that 30S IC-driven changes in the stability of IF2 and the conformational dynamics of the IF2•tRNA sub-complex regulate the efficiency and fidelity of subunit joining during translation initiation. Copyright © 2015. Published by Elsevier Ltd.
    Journal of Molecular Biology 01/2015; DOI:10.1016/j.jmb.2014.12.025 · 3.96 Impact Factor