title: Study of DNA binding and bending by Bacillus subtilis GabR, a PLP-dependent transcription factor.
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 10nM. 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.
  
  
  
  Characterization of the GabR DNA binding mode highlights the key role of DNA bending and interactions with bases outside the canonical direct repeats, and might be of general relevance for the action mechanism of MocR transcription factors.
authors.affiliation: Dipartimento di Bioscienze, Università degli Studi di Parma, Parma, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Amidani
authors.affiliation: Istituto di Biologia e Patologia Molecolari, Consiglio Nazionale delle Ricerche, Roma, Italy; Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Tramonti
authors.affiliation: Dipartimento di Farmacia, Università di Parma, Parma, Italy.
authors.forename: Andrea Valeria
authors.initials: AV
authors.lastname: Canosa
authors.affiliation: Dipartimento di Farmacia, Università di Parma, Parma, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Campanini
authors.affiliation: Dipartimento di Bioscienze, Università degli Studi di Parma, Parma, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Maggi
authors.affiliation: Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Italy.
authors.forename: Teresa
authors.initials: T
authors.lastname: Milano
authors.affiliation: Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Italy.
authors.forename: Martino L
authors.initials: ML
authors.lastname: di Salvo
authors.affiliation: Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Pascarella
authors.affiliation: Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli', Sapienza Università di Roma, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Contestabile
authors.affiliation: Dipartimento di Neuroscienze, Università di Parma, Parma, Italy; National Institute of Biostructures and Biosystems, Rome, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Bettati
authors.affiliation: Dipartimento di Bioscienze, Università degli Studi di Parma, Parma, Italy. Electronic address: claudio.rivetti@unipr.it.
authors.forename: Claudio
authors.initials: C
authors.lastname: Rivetti
mesh_terms: D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D000072417:Protein Domains; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D016415:Sequence Alignment; D013056:Spectrophotometry, Ultraviolet; D055672:Static Electricity; D014157:Transcription Factors / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D050505:Mutant Proteins; D014157:Transcription Factors; D005680:gamma-Aminobutyric Acid; D011732:Pyridoxal Phosphate
keywords: Atomic Force Microscopy; GABA metabolism; MocR; PLP enzyme; Transcription factor