PMID:9130723

Citation	Spurio, R, Falconi, M, Brandi, A, Pon, CL and Gualerzi, CO (1997) The oligomeric structure of nucleoid protein H-NS is necessary for recognition of intrinsically curved DNA and for DNA bending. EMBO J. 16:1795-805
Abstract	Escherichia coli hns, encoding the abundant nucleoid protein H-NS, was subjected to site-directed mutagenesis either to delete Pro115 or to replace it with alanine. Unlike the wild-type protein, hyperproduction of the mutant proteins did not inhibit macromolecular syntheses, was not toxic to cells and caused a less drastic compaction of the nucleoid. Gel shift and ligase-mediated circularization tests demonstrated that the mutant proteins retained almost normal affinity for non-curved DNA, but lost the wild-type capacity to recognize preferentially curved DNA and to actively bend non-curved DNA, a property of wild-type H-NS demonstrated here for the first time. DNase I foot-printing and in vitro transcription experiments showed that the mutant proteins also failed to recognize the intrinsically bent site of the hns promoter required for H-NS transcription autorepression and to inhibit transcription from the same promoter. The failure of the Pro115 mutant proteins to recognize curved DNA and to bend DNA despite their near normal affinity for non-curved DNA can be attributed to a defect in protein-protein interaction resulting in a reduced capacity to form oligomers observed in vitro and by a new in vivo test based on functional replacement by H-NS of the oligomerization domain (C-domain) of bacteriophage lambda cI repressor.
Links	PubMed PMC1169782 Online version:10.1093/emboj/16.7.1795
Keywords	Amino Acid Sequence; Bacterial Outer Membrane Proteins/chemistry; Bacterial Outer Membrane Proteins/genetics; Bacterial Outer Membrane Proteins/metabolism; Bacterial Proteins; Binding Sites; DNA/chemistry; DNA/metabolism; DNA Footprinting; DNA-Binding Proteins/chemistry; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Deoxyribonuclease I; Escherichia coli/genetics; Escherichia coli/metabolism; Kinetics; Macromolecular Substances; Mutagenesis, Site-Directed; Nucleic Acid Conformation; Oligodeoxyribonucleotides/chemistry; Protein Binding; Protein Structure, Secondary; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Sequence Deletion; Transcription, Genetic
edit table

Significance

You can help EcoliWiki by summarizing why this paper is useful

Useful Materials and Methods

You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.

Annotations

<annotationlinks/>

EcoliWiki Links

Add links to pages that link here (e.g. gene, product, method pages)

References

See Help:References for how to manage references in EcoliWiki.