Title
Study of DNA binding and bending by Bacillus subtilis GabR, a PLP-dependent transcription factor
Author
Davide Amidani, Angela Tramonti, Andrea Valeria Canosa, Barbara Campanini, Stefano Maggi, Teresa Milano, Martino L. di Salvo, Stefano Pascarella, Roberto Contestabile, Stefano Bettati, Claudio Rivetti
Year
2016
Journal
Biochimica et Biophysica Acta (BBA) - General Subjects
Abstract
GabR is a transcriptional regulator belonging to the MocR/GabR family, characterized by a N-terminal wHTH DNA-binding domain and a C-terminal effector binding and/or oligomerization domain, structurally homologous to aminotransferases (ATs). In the presence of γ-aminobutyrate (GABA) and pyridoxal 5′-phosphate (PLP), GabR activates the transcription of gabT and gabD genes involved in GABA metabolism. Here we report a biochemical and atomic force microscopy characterization of Bacillus subtilis GabR in complex with DNA. Complexes were assembled in vitro to study their stoichiometry, stability and conformation. The fractional occupancy of the GabR cognate site suggests that GabR binds as a dimer with Kd of 10 nM. Upon binding GabR bends the DNA by 80° as measured by anomalous electrophoretic mobility. With GABA we observed a decrease in affinity and conformational rearrangements compatible with a less compact nucleo-protein complex but no changes of the DNA bending angle. By employing promoter and GabR mutants we found that basic residues of the positively charged groove on the surface of the AT domain affect DNA affinity. The present data extend current understanding of the GabR-DNA interaction and the effect of GABA and PLP. A model for the GabR-DNA complex, corroborated by a docking simulation, is proposed.
Instrument
J-715
Keywords
Circular dichroism, Secondary structure, Protein denaturation, Thermal stability, Biochemistry