Mekkaoui Fadwa, Drewell Robert A, Dresch Jacqueline M, Spratt Donald E
Gustaf H. Carlson School of Chemistry and Biochemistry, Clark University, 950 Main Street, Worcester, MA 01610, United States of America.
Biology Department, Clark University, 950 Main Street, Worcester, MA 01610, United States of America.
Biochim Biophys Acta Gene Regul Mech. 2025 Mar;1868(1):195074. doi: 10.1016/j.bbagrm.2024.195074. Epub 2024 Dec 5.
Homeodomain transcription factors (TFs) bind to specific DNA sequences to regulate the expression of target genes. Structural work has provided insight into molecular identities and aided in unraveling structural features of these TFs. However, the detailed affinity and specificity by which these TFs bind to DNA sequences is still largely unknown. Qualitative methods, such as DNA footprinting, Electrophoretic Mobility Shift Assays (EMSAs), Systematic Evolution of Ligands by Exponential Enrichment (SELEX), Bacterial One Hybrid (B1H) systems, Surface Plasmon Resonance (SPR), and Protein Binding Microarrays (PBMs) have been widely used to investigate the biochemical characteristics of TF-DNA binding events. In addition to these qualitative methods, bioinformatic approaches have also assisted in TF binding site discovery. Here we discuss the advantages and limitations of these different approaches, as well as the benefits of utilizing more quantitative approaches, such as Mechanically Induced Trapping of Molecular Interactions (MITOMI), Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC), in determining the biophysical basis of binding specificity of TF-DNA complexes and improving upon existing computational approaches aimed at affinity predictions.
同源域转录因子（TFs）与特定的DNA序列结合，以调控靶基因的表达。结构研究为这些转录因子的分子特性提供了深入见解，并有助于揭示其结构特征。然而，这些转录因子与DNA序列结合的详细亲和力和特异性在很大程度上仍然未知。定性方法，如DNA足迹法、电泳迁移率变动分析（EMSA）、指数富集的配体系统进化（SELEX）、细菌单杂交（B1H）系统、表面等离子体共振（SPR）和蛋白质结合微阵列（PBMs），已被广泛用于研究TF-DNA结合事件的生化特性。除了这些定性方法外，生物信息学方法也有助于转录因子结合位点的发现。在这里，我们讨论这些不同方法的优点和局限性，以及利用更定量的方法，如机械诱导分子相互作用捕获（MITOMI）、微量热泳动（MST）和等温滴定量热法（ITC），在确定TF-DNA复合物结合特异性的生物物理基础以及改进现有针对亲和力预测的计算方法方面的益处。
