The Degree of Oligomerization of the H-NS Nucleoid Structuring Protein Is Related to Specific Binding to DNA

Cyril Badaut,Roy Williams,Véronique Arluison,Emeline Bouffartigues,Bruno Robert,Henri Buc,Sylvie Rimsky

doi:10.1074/jbc.m206037200

Abstract

At several E. coli promoters, initiation of transcription is repressed by a tight nucleoprotein complex formed by the assembly of the H-NS protein. In order to characterize the relationship between the structure of H-NS oligomers in solution and on relevant DNA fragments, we have compared wild-type H-NS and several transdominant H-NS mutants using gel shift assays, DNase I footprinting, analytical ultracentrifugation, and reactivity toward a cross-linking reagent. In solution, oligomerization occurs through two protein interfaces, one necessary to construct a dimeric core (and involving residues 1-64) and the other required for subsequent assembly of these dimers. We show that, as well as region 64-95, residues present in the NH(2)-terminal coiled coil domain also participate in this second interface. Our results support the view that the same interacting interfaces are also involved on the DNA. We propose that the dimeric core recognizes specific motifs, with the second interface being critical for their correct head to tail assembly. The COOH-terminal domain of the protein contains the DNA binding motif essential for the discrimination of this specific functional assembly over competitive nonspecific H-NS polymers.

Highlights

H-NS is a DNA-binding protein involved in structurally organizing the nucleoid of prokaryotic cells
Efficient repression requires first the building up of a substructure resulting from the binding of H-NS at the curved insert and recruitment by a cooperative process of H-NS molecules bound at other strategic sites and in particular at the Pribnow box of the gal control region
We used a gel retardation assay to characterize the ability of modified H-NS proteins to recognize specific DNA fragments [30, 34, 35]

Summary

EXPERIMENTAL PROCEDURES

Purification of H-NS Proteins—The different His6-tagged H-NS proteins were expressed in E. coli BL21␭DE3 as described by Williams et al [23]. The chemical cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) reacts with the carboxyl groups of glutamate or aspartate residues to produce an unstable O-acetyl-isourea, which in turn forms a reactive N-hydroxysuccinimide ester in the presence of N-hydroxysuccinimide (NHS) This intermediate undergoes a nucleophilic attack by the amine group of lysine residues, leading to the formation of covalent bonds between Glu (or Asp)/Lys amino acid pairs in close proximity to each other. Protein cross-linking reactions were performed at 100 ␮M protein concentration in a 40 mM HEPES buffer, pH 8, containing 8 mM magnesium aspartate, 60 mM potassium glutamate, 0.05% Nonidet P-40, and 2 mM dithiothreitol. Where I(t) is the relative intensity (in arbitrary units) of each band detected on the gel, kobs is the pseudo-first order rate of monomer disappearance (in minϪ1) during the reaction, and t is the time in minutes of the cross-linking reaction. Equilibrium sedimentation data were analyzed to yield weight average molecular masses using the program XL-A/XL-I data analysis software 4.0 supplied by Beckman

Electrophoretic Mobility Shift Assays

DNase I Footprint Experiments

Analytical Ultracentrifugation

DISCUSSION