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Journal of Bacteriology, June 2009, p. 3832-3841, Vol. 191, No. 12
0021-9193/09/$08.00+0 doi:10.1128/JB.01630-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Recruitment of the ParG Segregation Protein to Different Affinity DNA Sites
,
Massimiliano Zampini,1
Andrew Derome,1,
Simon E. S. Bailey,1,
Daniela Barillà,2 and
Finbarr Hayes1*
Faculty of Life Sciences and Manchester Interdisciplinary Biocentre, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom,1 Department of Biology, University of York, P.O. Box 373, York YO10 5YW, United Kingdom2
Received 17 November 2008/ Accepted 9 April 2009
The segrosome is the nucleoprotein complex that mediates accurate plasmid segregation. In addition to its multifunctional role in segrosome assembly, the ParG protein of multiresistance plasmid TP228 is a transcriptional repressor of the parFG partition genes. ParG is a homodimeric DNA binding protein, with C-terminal regions that interlock into a ribbon-helix-helix fold. Antiparallel β-strands in this fold are presumed to insert into the OF operator major groove to exert transcriptional control as established for other ribbon-helix-helix factors. The OF locus comprises eight degenerate tetramer boxes arranged in a combination of direct and inverted orientation. Each tetramer motif likely recruits one ParG dimer, implying that the fully bound operator is cooperatively coated by up to eight dimers. OF was subdivided experimentally into four overlapping 20-bp sites (A to D), each of which comprises two tetramer boxes separated by AT-rich spacers. Extensive interaction studies demonstrated that sites A to D individually are bound with different affinities by ParG (C > A 
B >> D). Moreover, comprehensive scanning mutagenesis revealed the contribution of each position in the site core and flanking sequences to ParG binding. Natural variations in the tetramer box motifs and in the interbox spacers, as well as in flanking sequences, each influence ParG binding. The OF operator apparently has evolved with sites that bind ParG dissimilarly to produce a nucleoprotein complex fine-tuned for optimal interaction with the transcription machinery. The association of other ribbon-helix-helix proteins with complex recognition sites similarly may be modulated by natural sequence variations between subsites.
* Corresponding author. Mailing address: Faculty of Life Sciences and Manchester Interdisciplinary Biocentre, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom. Phone: 44 161 3068934. Fax: 44 161 3065201. E-mail: finbarr.hayes{at}manchester.ac.uk
Published ahead of print on 17 April 2009.
Supplemental material for this article may be found at http://jb.asm.org/.
Present address: bioMerieux bv, Boseind 15, 5281 RM Boxtel, The Netherlands.
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Journal of Bacteriology, June 2009, p. 3832-3841, Vol. 191, No. 12
0021-9193/09/$08.00+0 doi:10.1128/JB.01630-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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