DNA binding properties of basic helix-loop-helix fusion proteins of Tal and E47.
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 x 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.
Ghosh
I
I
Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
Bishop
P
P
Chmielewski
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004586:Electrophoresis; D027283:Large Neutral Amino Acid-Transporter 1; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors
D017931:DNA Primers; D004268:DNA-Binding Proteins; D027283:Large Neutral Amino Acid-Transporter 1; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C000632119:Slc7a5 protein, rat; D051820:TCF Transcription Factors; C553060:Tcf7l1 protein, rat; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors