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

Renata Moreno1, Stefano Marzi, Pascale Romby

  • 1Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.

Nucleic Acids Research
|October 15, 2009
PubMed

Insights

The Crc protein in Pseudomonads regulates gene expression by binding to specific mRNA sequences, inhibiting translation. This study identifies the precise binding site on alkS mRNA, revealing how Crc controls catabolic pathways.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Gene Regulation

Background:

  • Crc (Carbon Regulation Control) is a global translational regulator in Pseudomonads.
  • It controls the induction hierarchy of various catabolic pathways.
  • Crc inhibits translation of specific mRNAs, like alkS and benR, in the presence of preferred carbon sources (amino acids).

Purpose of the Study:

  • To elucidate the binding site and mechanism of Crc interaction with target mRNAs.
  • To understand how Crc inhibits translation initiation.
  • To identify a consensus sequence for predicting new Crc targets.

Main Methods:

  • Secondary structure determination of alkS mRNA 5'-end using ribonucleases and chemical reagents.
  • Footprinting and band-shift assays with variant alkS mRNAs.
  • Localization of Crc-binding sites on alkS and benR mRNAs.

Main Results:

  • The secondary structure of the alkS mRNA 5'-end was determined.
  • Crc specifically binds to a short, unpaired, A-rich sequence near the alkS AUG start codon.
  • This binding prevents the formation of the translational initiation complex.
  • A similar binding site was found on benR mRNA.

Conclusions:

  • Crc directly inhibits translation by binding to specific mRNA sequences.
  • A consensus Crc-binding site sequence was derived, enabling prediction and validation of new Crc targets.
  • This work clarifies a key mechanism of translational regulation in Pseudomonads.

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