Background: 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. Methods: 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. Results: 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. Conclusions: 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. General Significance: 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.

Study of DNA binding and bending by Bacillus subtilis GabR, a PLP-dependent transcription factor / Amidani, Davide; Tramonti, Angela; Canosa, Andrea Valeria; Campanini, Barbara; Maggi, Stefano; Milano, Teresa; Di Salvo, Martino Luigi; Pascarella, Stefano; Contestabile, Roberto; Bettati, Stefano; Rivetti, Claudio. - In: BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS. - ISSN 0304-4165. - 1861:1(2017), pp. 3474-3489. [10.1016/j.bbagen.2016.09.013]

Study of DNA binding and bending by Bacillus subtilis GabR, a PLP-dependent transcription factor

AMIDANI, Davide;CANOSA, ANDREA VALERIA;CAMPANINI, Barbara;MAGGI, STEFANO;DI SALVO, MARTINO LUIGI;PASCARELLA, STEFANO;CONTESTABILE, ROBERTO;BETTATI, Stefano;RIVETTI, Claudio
2017

Abstract

Background: 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. Methods: 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. Results: 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. Conclusions: 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. General Significance: 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.
Study of DNA binding and bending by Bacillus subtilis GabR, a PLP-dependent transcription factor / Amidani, Davide; Tramonti, Angela; Canosa, Andrea Valeria; Campanini, Barbara; Maggi, Stefano; Milano, Teresa; Di Salvo, Martino Luigi; Pascarella, Stefano; Contestabile, Roberto; Bettati, Stefano; Rivetti, Claudio. - In: BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS. - ISSN 0304-4165. - 1861:1(2017), pp. 3474-3489. [10.1016/j.bbagen.2016.09.013]
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11381/2819673
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