The Crc global regulator binds to an unpaired A-rich motif at the Pseudomonas putida alkS mRNA coding sequence and inhibits translation initiation

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Abstract
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.