부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.jmb.2009.09.006 Byline: Martin Overgaard (1), Jonas Borch (1), Kenn Gerdes (2) Keywords: toxin; antitoxin; RelB; RelE; ribbon-helix-helix Abbreviations: RHH, ribbon-helix-helix; TA, toxin-antitoxin; WT, wild type; SPR, surface plasmon resonance; HMK, heart myosin kinase; X-gal, 5-bromo-4-chloro-3-indoyl-[beta]-d-galactoside; EDTA, ethylenediaminetetraacetic acid Abstract: RelB, the ribbon-helix-helix (RHH) repressor encoded by the relBE toxin-antitoxin locus of Escherichia coli, interacts with RelE and thereby counteracts the mRNA cleavage activity of RelE. In addition, RelB dimers repress the strong relBE promoter and this repression by RelB is enhanced by RelE; that is, RelE functions as a transcriptional co-repressor. RelB is a Lon protease substrate, and Lon is required both for activation of relBE transcription and for activation of the mRNA cleavage activity of RelE. Here we characterize the molecular interactions important for transcriptional control of the relBE model operon. Using an in vivo screen for relB mutants, we identified multiple nucleotide changes that map to important amino acid positions within the DNA-binding domain formed by the N-terminal RHH motif of RelB. Analysis of DNA binding of a subset of these mutant RHH proteins by gel-shift assays, transcriptional fusion assays and a structure model of RelB-DNA revealed amino acid residues making crucial DNA-backbone contacts within the operator (relO) DNA. Mutational and footprinting analyses of relO showed that RelB dimers bind on the same face of the DNA helix and that the RHH motif recognizes four 6-bp repeats within the bipartite binding site. The spacing between each half-site was found to be essential for cooperative interactions between adjacently bound RelB dimers stabilized by the co-repressor RelE. Kinetic and stoichiometric measurements of the interaction between RelB and RelE confirmed that the proteins form a high-affinity complex with a 2:1 stoichiometry. Lon degraded RelB in vitro and degradation was inhibited by RelE, consistent with the proposal that RelE protects RelB from proteolysis by Lon in vivo. Author Affiliation: (1) Department of Biochemistry and Molecular Biology, University of Southern Denmark Odense, Campusvej 55, 5230 Odense M, Denmark (2) Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle NE2 4HH, UK Article History: Received 27 May 2009; Revised 31 August 2009; Accepted 1 September 2009 Article Note: (miscellaneous) Edited by R. Ebright