Single-molecule DNA unzipping reveals asymmetric modulation of a transcription factor by its binding site sequence and context

• Published in: Nucleic acids research Vol. 46; no. 3; pp. 1513 - 1524
• Main Authors: Rudnizky, Sergei, Khamis, Hadeel, Malik, Omri, Squires, Allison H, Meller, Amit, Melamed, Philippa, Kaplan, Ariel
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 16.02.2018
• Subjects: Structural Biology
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkx1252

Abstract Most functional transcription factor (TF) binding sites deviate from their 'consensus' recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used single-molecule DNA unzipping with optical tweezers to study how Egr-1, a TF harboring three zinc fingers (ZF1, ZF2 and ZF3), is modulated by the sequence and context of its functional sites in the Lhb gene promoter. We find that both the core 9 bp bound to Egr-1 in each of the sites, and the base pairs flanking them, modulate the affinity and structure of the protein-DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking ZF3. Characterization of the dissociation time of Egr-1 revealed that a local, mechanical perturbation of the interactions of ZF3 destabilizes the complex more effectively than a perturbation of the ZF1 interactions. Our results reveal a novel role for ZF3 in the interaction of Egr-1 with other proteins and the DNA, providing insight on the regulation of Lhb and other genes by Egr-1. Moreover, our findings reveal the potential of small changes in DNA sequence to alter transcriptional regulation, and may shed light on the organization of regulatory elements at promoters.