Effects of Complex Formation on Motion in DNA Binding Domains of ARFs

Abstract

The auxin response factor (ARF) family is a set of DNA binding transcription factors found in the model plant Arabidopsis thaliana in addition to many other food and fuel crops. Along with two other frequently conserved domains, all ARFs contain an N‐terminus DNA binding domain (DBD) that includes a B3 DNA binding motif and a dimerization subdomain. While the structure of example DBDs have been solved, motion within this domain is poorly understood. The interplay of these domains may provide selectivity in binding to variants of target DNA constructs. Our work examines the DBDs of ARF1 and ARF5, a transcription repressor and transcription activator respectively, as ARF5 is known to be more permissive than ARF1 to a larger spacing between binding sites on DNA. Our work directly tests the hypothesis that the ability to bind to a broader range of DNA targets relates to observable differences in structural flexibility. We combine spectroscopic and chromatographic methods of resolving both complex formation and molecular motion for different combinations of target DNA and DBD. We utilize fluorescence spectroscopy as it resolves protein motions and associations that occur on the same timescale as the fluorescent lifetimes (~ns). Labeled proteins can also be studied at low physiological concentrations and within an aqueous buffer. We optimized the expression and purification of the protein as well as two alternative fluorophore conjugation methods to compare thiol‐reactive fluorophore labeling to a newer technique that targets the N‐terminus. Under conditions of known dissociated versus associated states, we apply both steady‐state and time‐resolved fluorescence anisotropy to sense rotational correlation times of these proteins and distinguish subdomain motions in the presence and absence of DNA. Our work demonstrates that the N‐terminal probes are more sensitive to molecular changes likely due to its placement at the hinge between the DNA binding domain and the dimerization domain. Consistent with our hypothesis, ARF5 shifts from a highly dynamic state to a more rigid state upon complex formation while ARF1 remains more unperturbed. These differences in the available structural ensemble provide valuable insights into the structural basis of target specificity as well as the interplay of these transcriptional on‐ and off‐switches.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.