PMID:9398522

Citation	Ueguchi, C, Seto, C, Suzuki, T and Mizuno, T (1997) Clarification of the dimerization domain and its functional significance for the Escherichia coli nucleoid protein H-NS. J. Mol. Biol. 274:145-51
Abstract	The Escherichia coli nucleoid protein, H-NS, functions as a global regulator for expression of a wide variety of genes. We recently analyzed the structure-function relationship of H-NS with special reference to the domains responsible for transcriptional repression and DNA-binding, respectively. However, identification of the presumed dimerization domain of H-NS and its functional significance was elusive. To address this particular issue, we first examined a set of N-terminally or C-terminally truncated forms of H-NS, in terms of their so-called dominant-negative effect on the in vivo function of the wild-type H-NS. The results showed that certain truncated forms exhibit such a dominant-negative effect, but others did not. As judged by the results of the dominant-negative effect, it was assumed that a relatively central portion of H-NS extending from residues 21 to 63 is involved in dimerization. This was confirmed by an in vitro chemical cross-linking analysis and a gel filtration analysis with these truncated forms of H-NS. Furthermore, the use of the dominant-negative phenotype, caused by a truncated form of H-NS (named N91), allowed us to isolate a missense mutant, which was expected to be specifically defective in dimerization. This mutant had an amino acid substitution at position 30 (Leu30 to Pro) in N91 consisting of the N-terminal 91 amino acids of H-NS. This mutant was indeed defective in the in vitro ability to form a heterodimer with the wild-type H-NS. When this particular single amino acid substitution was introduced into the full-length H-NS, the resultant H-NS mutant had lost the ability to form dimers in vitro and to function as a transcriptional repressor. These findings collectively provided us with evidence that the ability of H-NS to form a dimer is crucial for H-NS to function as a transcriptional repressor.
Links	PubMed Online version:10.1006/jmbi.1997.1381
Keywords	Amino Acid Sequence; Bacterial Proteins; Blotting, Western; Cross-Linking Reagents/metabolism; DNA, Bacterial/metabolism; DNA-Binding Proteins/chemistry; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Dimerization; Dimethyl Suberimidate/metabolism; Escherichia coli/chemistry; Escherichia coli/genetics; Gene Expression Regulation, Bacterial; Lac Operon/genetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Plasmids; Repressor Proteins/chemistry; Repressor Proteins/genetics; Repressor Proteins/metabolism; Sequence Deletion; Transcription, Genetic; Transfection; beta-Galactosidase/genetics; beta-Galactosidase/metabolism
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