E. coli DNA associated with isolated Hfq interacts with Hfq's distal surface and C-terminal domain

Taylor B. Updegrove, John J. Correia, Roberto Galletto, Wlodzimierz Bujalowski, Roger M. Wartell

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

The RNA-binding protein Hfq has been studied extensively for its function as a modulator of gene expression at the post-transcriptional level. While most Hfq studies have focused on the protein's interaction with sRNAs and mRNAs, Hfq binding to DNA has been observed but is less explored. During the isolation of Hfq from Escherichia coli, we found genomic DNA fragments associated with the protein after multiple steps of purification. Sequences of 41 amplified segments from the DNA fragments associated with Hfq were determined. A large fraction of the DNA segments were predicted to have significant helical axis curvature and were from genes associated with membrane proteins, characteristics unexpected for non-specific binding. Analysis by analytical ultracentrifugation indicated that rA18 binding to Hfq disrupts Hfq-DNA interactions. The latter observation suggests Hfq binding to DNA involves its distal surface. This was supported by a gel mobility shift assay that showed single amino acid mutations on the distal surface of Hfq inhibited Hfq binding to duplex DNA, while six of seven mutations on the proximal surface and outer circumference of the hexamer did not prevent Hfq binding. Two mutated Hfq which have portions of their C-terminal domain removed also failed to bind to DNA. The apparent Kd for binding wild type Hfq to several duplex DNA was estimated from a gel mobility shift assay to be ~400nM.

Original languageEnglish (US)
Pages (from-to)588-596
Number of pages9
JournalBiochimica et Biophysica Acta - Gene Regulatory Mechanisms
Volume1799
Issue number8
DOIs
StatePublished - Aug 2010

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Escherichia coli
DNA
Electrophoretic Mobility Shift Assay
Assays
Gels
Mutation
RNA-Binding Proteins
Ultracentrifugation
Gene expression
Modulators
Purification
Membrane Proteins
Proteins
Genes
Observation
Gene Expression
Amino Acids
Messenger RNA

Keywords

  • Hfq protein
  • Hfq-DNA binding

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Genetics
  • Molecular Biology
  • Structural Biology
  • Medicine(all)

Cite this

E. coli DNA associated with isolated Hfq interacts with Hfq's distal surface and C-terminal domain. / Updegrove, Taylor B.; Correia, John J.; Galletto, Roberto; Bujalowski, Wlodzimierz; Wartell, Roger M.

In: Biochimica et Biophysica Acta - Gene Regulatory Mechanisms, Vol. 1799, No. 8, 08.2010, p. 588-596.

Research output: Contribution to journalArticle

Updegrove, Taylor B. ; Correia, John J. ; Galletto, Roberto ; Bujalowski, Wlodzimierz ; Wartell, Roger M. / E. coli DNA associated with isolated Hfq interacts with Hfq's distal surface and C-terminal domain. In: Biochimica et Biophysica Acta - Gene Regulatory Mechanisms. 2010 ; Vol. 1799, No. 8. pp. 588-596.
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