Mechanism of hcnA mRNA recognition in the Gac/Rsm signal transduction pathway of Pseudomonas fluorescens

Mol Microbiol. 2007 Oct;66(2):341-56. doi: 10.1111/j.1365-2958.2007.05909.x. Epub 2007 Sep 10.

Abstract

In the plant-beneficial bacterium Pseudomonas fluorescens CHA0, the expression of antifungal exoproducts is controlled by the GacS/GacA two-component system. Two RNA binding proteins (RsmA, RsmE) ensure effective translational repression of exoproduct mRNAs. At high cell population densities, GacA induces three small RNAs (RsmX, RsmY, RsmZ) which sequester both RsmA and RsmE, thereby relieving translational repression. Here we systematically analyse the features that allow the RNA binding proteins to interact strongly with the 5' untranslated leader mRNA of the P. fluorescens hcnA gene (encoding hydrogen cyanide synthase subunit A). We obtained evidence for three major RsmA/RsmE recognition elements in the hcnA leader, based on directed mutagenesis, RsmE footprints and toeprints, and in vivo expression data. Two recognition elements were found in two stem-loop structures whose existence in the 5' leader region was confirmed by lead(II) cleavage analysis. The third recognition element, which overlapped the hcnA Shine-Dalgarno sequence, was postulated to adopt either an open conformation, which would favour ribosome binding, or a stem-loop structure, which may form upon interaction with RsmA/RsmE and would inhibit access of ribosomes. Effective control of hcnA expression by the Gac/Rsm system appears to result from the combination of the three appropriately spaced recognition elements.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • Binding Sites / genetics
  • Electrophoretic Mobility Shift Assay
  • Gene Expression Regulation, Bacterial
  • Molecular Sequence Data
  • Mutation
  • Oxidoreductases Acting on CH-NH2 Group Donors / genetics*
  • Oxidoreductases Acting on CH-NH2 Group Donors / metabolism*
  • Protein Binding
  • Pseudomonas fluorescens / genetics
  • Pseudomonas fluorescens / metabolism*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • RNA-Binding Proteins / physiology
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction / genetics
  • Signal Transduction / physiology*
  • beta-Galactosidase / genetics
  • beta-Galactosidase / metabolism

Substances

  • Bacterial Proteins
  • GacA protein, Bacteria
  • RNA, Messenger
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • Oxidoreductases Acting on CH-NH2 Group Donors
  • glycine dehydrogenase (cyanide-forming)
  • beta-Galactosidase