The major dimerization determinants of the nitrogen regulatory protein NTRC from enteric bacteria lie in its carboxy-terminal domain

Karl E. Klose, Anne K. North, Kenneth M. Stedman, Sydney Kustu

Research output: Contribution to journalArticle

56 Scopus citations

Abstract

The NTRC protein (nitrogen regulatory protein C) of enteric bacteria is an enhancer-binding protein that activates transcription by the σ54-holoenzyme form of RNA polymerase. NTRC is a homodimeric protein that binds to a dyad-symmetrical site in DNA. To activate transcription NTRC must be phosphorylated and must form an and propriate oligomeric species at an enhancer. In order to study subunit exchange between NTRC dimers, we constructed a fusion of the maltose-binding protein (MBP) to the amino-terminal end of NTRC (MBP-NTRC) and visualized the formation of heterodimers between MBP-NTP, C and wild-type NTRC by a gel-mobility shift assay for DNA-binding. When MBP-NTRC is mixed with wild-type NTRG at 37°C, subunit exchange occurs rapidly. The apparent half-life for dissociation of homodimers of NTRC is two to three minutes at 37 °C and is not changed by phosphorylation. The isolated carboxy-terminal domain of NTRC (91 amino acid residues) forms heterodimers with both wild-type NTRC and MBP-NTRC, indicating that the C-terminal domain is sufficient for dimerization. The apparent rate of dissociation of homodimers of the C-terminal domain is essentially the same as that of full-length NTRC, indicating that the major dimerization determinants of the protein lie in its C-terminal domain. Congruent with this, a truncated form of NTRC from which the last 58 amino acid residues were removed is a monomer in solution. Moreover, truncated forms of NTPIC from which the last 16 or 26 amino acid residues were removed are predominantly monomeric in solution, as is a mutant form with the amino acid substitution A410E in its C-terminal domain. Monomerization of the above mutant forms of NTRC can be rationalized on the basis of homology between the C-terminal region of NTRC and a 50 amino acid residue region of the factor for inversion stimulation (FIS) protein.

Original languageEnglish (US)
Pages (from-to)233-245
Number of pages13
JournalJournal of Molecular Biology
Volume241
Issue number2
DOIs
StatePublished - Aug 11 1994
Externally publishedYes

Keywords

  • DNA-binding
  • FIS protein
  • Phosphorylation
  • Protein purification
  • Transcriptional activation

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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