Specific binding between Arabidopsis thaliana phytochrome-interacting factor 3 (AtPIF3) bHLH and G-box originated prior to embryophyte emergence
Journal
BMC Plant Biology
Journal Volume
24
Journal Issue
1
ISSN
1471-2229
Date Issued
2024-11-11
Author(s)
DOI
10.1186/s12870-024-05777-z
Abstract
The basic helix-loop-helix (bHLH) domain via critical amino acid residues on basic region binding to E-box (5′-CANNTG-3′) is known in embryophyte. However, the dictated E-box types selection by bHLH dimers and the significant impact of these critical amino acid residues along embryophyte evolution remain unclear. The Arabidopsis thaliana PIF3-bHLH (AtPIF3-bHLH) recombinant protein and a series of AtPIF3-bHLH mutants were synthesized and analyzed. The reduced DNA binding ability and affinity of AtPIF3-bHLH point-mutation proteins, observed via fluorescence-based electrophoretic mobility shift assay (fEMSA) and isothermal titration calorimetry (ITC), suggest the critical role of these DNA-recognition sites in maintaining the AtPIF3-bHLH–DNA interaction. The purifying selection signals and the DNA-recognition-site conservation at the species level suggest the invariant roles of these sites throughout embryophyte evolution. The G-box outcompeted other types of E-box for binding in our competitive fEMSAs. The dynamic hydrogen bond formed between AtPIF3-bHLH and the G-box core indicates flexible identification of the core region. These features highlight a fast fixation of the bHLH-G-box recognition mechanism through embryophyte evolution and serve as a blueprint for studying DNA recognition determinants of other TF families.
Subjects
Circadian clock
Conservation
DNA binding preference
Protein-DNA interaction
Publisher
Springer Science and Business Media LLC
Description
Article number 1060
Type
journal article