Recruitment of the ParG segregation protein to different affinity DNA sites.
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 beta-strands in this fold are presumed to insert into the O(F) operator major groove to exert transcriptional control as established for other ribbon-helix-helix factors. The O(F) 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. O(F) 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 approximately 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 O(F) 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.
Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
Massimiliano
M
Zampini
Andrew
A
Derome
Simon E S
SE
Bailey
Daniela
D
Barillà
Finbarr
F
Hayes
D051103:1-Acylglycerol-3-Phosphate O-Acyltransferase / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009875:Operator Regions, Genetic; D010957:Plasmids / Q000235:genetics*; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C504447:ParG protein, E coli; D012097:Repressor Proteins; D051103:1-Acylglycerol-3-Phosphate O-Acyltransferase; C495091:plsC protein, E coli