Báo cáo khoa học: Characterization of HbpR binding by site-directed mutagenesis of its DNA-binding site and by deletion of the effector domain

Characterization of HbpR binding by site-directed mutagenesis of its DNA-binding site and by deletion of the effector domain

Tác giả: David Tropel and Jan R. van der Meer

Lĩnh vực: Environmental Microbiology and Molecular Ecotoxicology, Fundamental Microbiology

Nội dung tài liệu: This study investigates the binding interactions of the enhancer binding protein HbpR with its DNA operator region, specifically focusing on the hbpC promoter in Pseudomonas azelaica. Through site-directed mutagenesis of the operator and DNA-binding assays, the research elucidates critical nucleotides and structural motifs involved in HbpR binding. The study also examines the role of the N-terminal sensing A-domain of HbpR in DNA binding and transcriptional activation. Findings suggest that while the A-domain is crucial for activation, the protein can still bind DNA and form oligomeric complexes in its absence. The research proposes a model for HbpR-DNA complex formation, highlighting the importance of specific interactions and the potential for self-association.

Mục lục chi tiết:

• Keywords
• Correspondence
• *Present address
• Introduction
• Results
• Mutagenesis of the HbpR-binding sites
• DNasel footprinting analysis
• Construction of a AA HbpR derivative
• DNA-binding characteristics of the ∆A-HbpR protein
• Activation of the hbpC promoter by CBP-AA-HbpR
• Discussion
• Experimental procedures
• Strains and medium
• Recombinant DNA techniques
• Mutations of UASS C-1/C-2
• Cloning of the AA-hbpR expression vector
• In vivo ∆A-HbpR activation
• Purification of HbpR fusion protein, EMSAS and DNasel footprinting
• Supplementary material