Protocols for protein-DNA binding analysis of a zinc finger transcription factor bound to its cognate promoter

Lana C Ly; Yang Yang; Fudong Li; Merlin Crossley; Yunyu Shi; Kate G R Quinlan

doi:10.1016/j.xpro.2022.101598

Protocols for protein-DNA binding analysis of a zinc finger transcription factor bound to its cognate promoter

STAR Protoc. 2022 Jul 31;3(3):101598. doi: 10.1016/j.xpro.2022.101598. eCollection 2022 Sep 16.

Authors

Lana C Ly¹, Yang Yang², Fudong Li³, Merlin Crossley⁴, Yunyu Shi⁵, Kate G R Quinlan⁶

Affiliations

¹ School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia. Electronic address: lana.ly@unsw.edu.au.
² School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, China; Ministry of Education Key Laboratory for Membraneless Organelles and Cellular Dynamics, University of Science and Technology of China, Hefei, China.
³ Ministry of Education Key Laboratory for Membraneless Organelles and Cellular Dynamics, University of Science and Technology of China, Hefei, China.
⁴ School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
⁵ School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, China; Ministry of Education Key Laboratory for Membraneless Organelles and Cellular Dynamics, University of Science and Technology of China, Hefei, China. Electronic address: yyshi@ustc.edu.cn.
⁶ School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia. Electronic address: kate.quinlan@unsw.edu.au.

Abstract

Here, we describe protocols to interrogate the binding of the zinc fingers of the transcription factor ZBTB7A to the fetal γ-globin (HBG) promoter. We detail the steps for performing electrophoretic mobility shift assays (EMSAs), X-ray crystallography, and isothermal titration calorimetry (ITC) to explore this interaction. These techniques could readily be applied to the structural studies of other zinc finger transcription factors and cognate DNA sequences. For complete details on the use and execution of this protocol, please refer to Yang et al. (2021).

Keywords: Biophysics; Molecular Biology; Protein Biochemistry; Protein expression and purification; X-ray Crystallography.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Line, Tumor
DNA / genetics
DNA-Binding Proteins* / genetics
Transcription Factors* / genetics
Zinc Fingers

Substances

DNA-Binding Proteins
Transcription Factors
DNA