title: Probing the relation between protein-protein interactions and DNA binding for a linker mutant of the bacterial nucleoid protein H-NS.
abstract: We have investigated the relationship between oligomerization in solution and DNA binding for the bacterial nucleoid protein H-NS. This was done by comparing oligomerization and DNA binding of H-NS with that of a H-NS D68V-D71V linker mutant. The double linker mutation D68V-D71V, that makes the linker significantly more hydrophobic, leads to a dramatically enhanced and strongly temperature-dependent H-NS oligomerization in solution, as detected by dynamic light scattering. The DNA binding affinity of H-NS D68V-D71V for the hns promoter region is lower and has stronger temperature dependence than that of H-NS. DNase I footprinting experiments show that at high concentrations, regions protected by H-NS D68V-D71V are larger and less defined than for H-NS. In vitro transcription assays show that the enhanced protection also leads to enhanced transcriptional repression. Whereas the lower affinity of the H-NS D68V-D71V for DNA could be caused by competition between oligomerization in solution and oligomerization on DNA, the larger size of protected regions clearly confirms the notion that cooperative binding of H-NS to DNA is related to protein-protein interactions. These results emphasize the relative contributions of protein-protein interactions and substrate-dependent oligomerization in the control of gene repression operated by H-NS.
authors.affiliation: University of Camerino, School of Biosciences and Biotechnologies, 62032 Camerino, MC, Italy.
authors.forename: Mara
authors.initials: M
authors.lastname: Giangrossi
authors.affiliation: Lab. Phys. Chem. Colloid Sci., Wageningen University, Dreijenplein 4, 6703 HB Wageningen, The Netherlands.
authors.forename: Kathelijne
authors.initials: K
authors.lastname: Wintraecken
authors.affiliation: University of Camerino, School of Biosciences and Biotechnologies, 62032 Camerino, MC, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Spurio
authors.affiliation: Lab. Phys. Chem. Colloid Sci., Wageningen University, Dreijenplein 4, 6703 HB Wageningen, The Netherlands. Electronic address: renko.devries@wur.nl.
authors.forename: Renko
authors.initials: R
authors.lastname: de Vries
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D050505:Mutant Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D055503:Protein Multimerization
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; D050505:Mutant Proteins
keywords: DNA-binding protein; Protein–protein interaction; Transcription regulation