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

In the presence of 2-hydroxybiphenyl, the enhancer binding protein, HbpR, activates ther54-dependent PhbpC promoter and controls the initial steps of 2-hydroxybiphenyl degradation inPseudomonas azelaica. In the activa-tion process, an oligomeric HbpR complex of unknown subunit composi-tion binds to an operator region containing two imperfect palindromic sequences. Here, the HbpR–DNA binding interactions were investigated by site-directed mutagenesis of the operator region and by DNA-binding assays using purified HbpR. . | iFEBS Journal Characterization of HbpR binding by site-directed mutagenesis of its DNA-binding site and by deletion of the effector domain David Tropel1 and Jan R. van der Meer1 2 1 Process of EnvironmentalMicrobiology and Molecular Ecotoxicology Swiss Federallnstitute for EnvironmentalScience and Technology EAWAG Dubendorf Switzerland 2 Department of FundamentalMicrobiology University of Lausanne Switzerland Keywords Pseudomonas azelaica o54-dependent transcriptionalactivators XylR family Correspondence J. R. van der Meer Department of FundamentalMicrobiology Batiment de Biologie University of Lausanne CH 1015 Lausanne Switzerland Fax 41 21 6925605 Tel 41 21 6925630 E-mail Present address M. E. Muller Institut-Biozentrum Klingelbergstrasse 70 CH-4056 Basel Switzerland Received 25 November 2004 revised 27 January 2005 accepted 10 February 2005 doi In the presence of 2-hydroxybiphenyl the enhancer binding protein HbpR activates the r54-dependent PhbpC promoter and controls the initial steps of 2-hydroxybiphenyl degradation in Pseudomonas azelaica. In the activation process an oligomeric HbpR complex of unknown subunit composition binds to an operator region containing two imperfect palindromic sequences. Here the HbpR-DNA binding interactions were investigated by site-directed mutagenesis of the operator region and by DNA-binding assays using purified HbpR. Mutations that disrupted the twofold symmetry in the palindromes did not affect the binding affinity of HbpR but various mutations along a 60 bp region and also outside the direct palindromic sequences decreased the binding affinity. Footprints of HbpR on mutant operator fragments showed that a partial loss of binding contacts occurs suggesting that the binding of one HbpR protomer in the oligomeric complex is impaired whilst leaving the other contacts intact. An HbpR variant devoid of its N-terminal sensing A-domain was unable to activate .

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