Journal
NUCLEIC ACIDS RESEARCH
Volume 37, Issue 22, Pages 7678-7690Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkp825
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Funding
- European Molecular Biology Organization [ASTF 224.00-2008]
- Spanish Ministry of Science and Innovation [BFU2006-00767/BMC, BFU2009-07009/BMC]
- Instituto de Salud Carlos III [REIPI RD06/0008]
- Centre National de la Recherche Scientifique (CNRS)
- French National Agency for Research [ANR-07-blan-0351-02]
- Agence Nationale de la Recherche (ANR) [ANR-07-BLAN-0351] Funding Source: Agence Nationale de la Recherche (ANR)
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Crc is a key global translational regulator in Pseudomonads that orchestrates the hierarchy of induction of several catabolic pathways for amino acids, sugars, hydrocarbons or aromatic compounds. In the presence of amino acids, which are preferred carbon sources, Crc inhibits translation of the Pseudomonas putida alkS and benR mRNAs, which code for transcriptional regulators of genes required to assimilate alkanes (hydrocarbons) and benzoate (an aromatic compound), respectively. Crc binds to the 5'-end of these mRNAs, but the sequence and/or structure recognized, and the way in which it inhibits translation, were unknown. We have determined the secondary structure of the alkS mRNA 5'-end through its sensitivity to several ribonucleases and chemical reagents. Footprinting and band-shift assays using variant alkS mRNAs have shown that Crc specifically binds to a short unpaired A-rich sequence located adjacent to the alkS AUG start codon. This interaction is stable enough to prevent formation of the translational initiation complex. A similar Crc-binding site was localized at benR mRNA, upstream of the Shine-Dalgarno sequence. This allowed predicting binding sites at other Crc-regulated genes, deriving a consensus sequence that will help to validate new Crc targets and to discriminate between direct and indirect effects of this regulator.
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