期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 390, 期 5, 页码 1019-1029出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2009.06.002
关键词
DNA binding; HucR; transcriptional regulator; tryptophan fluorescence; uric acid
资金
- National Science Foundation [MCB-0414875, MCB-0744240]
- LSU Faculty Research Grant
Members of the multiple antibiotic resistance regulator (MarR) family control gene expression in a variety of metabolic processes in bacteria and archaea. Hypothetical uricase regulator (HucR), which belongs to the ligand-responsive branch of the MarR family, regulates uricase expression in Deinococcus radiodurans by binding a shared promoter region between uricase and HucR genes. We show here that HucR responds only to urate and, to a lesser extent, to xanthine by attenuated DNA binding, compared to other intermediates of purine degradation. Using molecular-dynamics-guided mutational analysis, we identified the ligand-binding site in HucR. Electrophoretic mobility shift assays and intrinsic Trp fluorescence have identified W20 from the N-terminal helix and R80 from helix 3, which serves as a scaffold for the DNA recognition helix, as being essential for ligand binding. Using structural data combined with in silico and in vitro analyses, we propose a mechanism for the attenuation of DNA binding in which a conformational change initiated by charge repulsion due to a bound ligand propagates to DNA recognition helices. This mechanism may apply generally to MarR homologs that bind anionic phenolic ligands. (C) 2009 Elsevier Ltd. All rights reserved.
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