Phosphorylation-dependent conformational changes in OmpR, an osmoregulatory DNA-binding protein of Escherichia coli

Linda Kenney, Mark D. Bauer, Thomas J. Silhavy

Research output: Contribution to journalArticle

79 Citations (Scopus)

Abstract

Osmoregulated porin gene expression in Escherichia coli is controlled by the two-component regulatory system EnvZ and OmpR. EnvZ, the osmosensor, is an inner membrane protein and a histidine kinase. EnvZ phosphorylates OmpR, a cytoplasmic DNA-binding protein, on an aspartyl residue. Phospho-OmpR binds to the promoters of the porin genes to regulate the expression of ompF and ompC. We describe the use of limited proteolysis by trypsin and ion spray mass spectrometry to characterize phospho-OmpR and the conformational changes that occur upon phosphorylation. Our results are consistent with a two- domain structure for OmpR, an N-terminal phosphorylation domain joined to a C-terminal DNA-binding domain by a flexible linker region. In the presence of acetyl phosphate, OmpR is phosphorylated at only one site. Phosphorylation induces a conformational change that is transmitted to the C-terminal domain via the central linker. Previous genetic analysis identified a region in the C-terminal domain that is required for transcriptional activation. Our results indicate that this region is within a surface-exposed loop. We propose that this loop contacts the α subunit of RNA polymerase to activate transcription. Mass spectrometry also reveals an unusual dephosphorylated form of OmpR, the potential significance of which is discussed.

Original languageEnglish (US)
Pages (from-to)8866-8870
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume92
Issue number19
DOIs
StatePublished - Sep 12 1995
Externally publishedYes

Fingerprint

DNA-Binding Proteins
Porins
Phosphorylation
Escherichia coli
Mass Spectrometry
DNA-Directed RNA Polymerases
Trypsin
Transcriptional Activation
Proteolysis
Membrane Proteins
Ions
Gene Expression
DNA
Genes

Keywords

  • ion spray mass spectrometry
  • response regulators
  • signal transduction
  • transcriptional activation
  • two-component regulatory system

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Phosphorylation-dependent conformational changes in OmpR, an osmoregulatory DNA-binding protein of Escherichia coli. / Kenney, Linda; Bauer, Mark D.; Silhavy, Thomas J.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 92, No. 19, 12.09.1995, p. 8866-8870.

Research output: Contribution to journalArticle

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