Interactions Between Intrinsically Disordered Domains of Nuclear Receptors and DNA Studied with Single‐Molecule Optical Tweezers, Computational Simulations, and Cell Assays

Abstract

Nuclear hormone receptors (NR) are transcription factors that relay cellular signals through distinct multiprotein assemblies. Hormonal signals produce structural changes within NRs that determine the composition of the interacting proteins. NRs are characteristically modular proteins. At the N terminus is an intrinsically disordered N-terminal domain (NTD) followed by a DNA-binding domain (DBD). The DBD recognizes DNA sites at the promoter of specific genes. At the C terminus is the ligand-binding domain (LBD) which also contains a dimerization interface. Agonist binding to the LBD results in conformational changes associated with a transcriptionally active state where the LBD rearranges to create a docking site for transcriptional regulator proteins such as the steroid receptor coactivator 1 (SRC1). Whereas the general events involved in NR-mediated gene transcription are well understood, fundamental issues remain unsolved. For instance, details of how the NR transcription factors recognize DNA sequences near the transcribed gene, so acting to ensure the optimal assembly of proteins actively involved in transcription, are still unknown. With the thyroid hormone receptor-α (TRα) as a model system, our studies are aimed to uncover how NRs recognize specific DNA sites to secure optimal gene transcription. To achieve our objectives, we have employed a combination of structural biophysics and in cellulo assays. We utilize single-molecule microscopy using optical tweezers, computational simulations of molecular dynamics and cell-based transcription activity assays. With these techniques, we characterize how DNA sequences distant from the canonical binding site of TRα can regulate transcriptional activity.