DNA binding properties of basic helix-loop-helix fusion proteins of Tal and E47

I. Ghosh, P. Bishop, J. Chmielewski

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

The basic helix-loop-helix (bHLH) transcription factor Tal has been shown to form heterodimers with the ubiquitously expressed bHLH transcription factor E47 and thereby modulate gene expression. The absence of homodimeric Tal-DNA complexes had been attributed to the inability of Tal to homodimerize, but subsequent studies have shown that the bHLH region of Tal does homodimerize. In order to correlate the contributions of both the basic region and the helix-loop-helix (HLH) domain to the lack of DNA binding by Tal homodimers, mutant and fusion proteins based on Tal and E47 were designed and synthesized. Size-exclusion chromatography established that all mutant and fusion proteins were dimeric. Point mutations were made within the basic region of Tal based on residues within E47 that are essential for DNA binding, but an affinity for DNA was not observed. Even complete replacement of the basic region in Tal with the basic region of E47, in an E47-Tal fusion protein, did not confer DNA binding upon the protein. However, when the dimerization domain in Tal was replaced with its E47 counterpart, in a Tal-E47 fusion protein, sequence specific DNA binding was observed with an apparent dissociation constant of 3.6×10-9 M2. Furthermore, circular dichroism studies showed that the basic region of Tal in the Tal-E47 fusion protein underwent a random coil to helix transition in the presence of a specific DNA probe. These experimental observations indicate that the inability of Tal homodimers to recognize DNA stems from a misalignment of its basic region with respect to the HLH domain, rather than an intrinsic inability of the Tal basic region to bind DNA.

Original languageEnglish (US)
Pages (from-to)354-360
Number of pages7
JournalJournal of Peptide Research
Volume57
Issue number5
DOIs
StatePublished - Aug 31 2001
Externally publishedYes

Keywords

  • Basic helix-loop-helix
  • DNA binding
  • Dimerization
  • E47
  • Fusion proteins
  • Tal
  • Transcription factor

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology

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