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Sequence-specific Recognition of DNA by the C-terminal Domain of Nucleoid-associated Protein H-NS
- Source :
- Journal of Biological Chemistry. 284:30453-30462
- Publication Year :
- 2009
- Publisher :
- Elsevier BV, 2009.
-
Abstract
- The molecular determinants necessary and sufficient for recognition of its specific DNA target are contained in the C-terminal domain (H-NSctd) of nucleoid-associated protein H-NS. H-NSctd protects from DNaseI cleavage a few short DNA segments of the H-NS-sensitive hns promoter whose sequences closely match the recently identified H-NS consensus motif (tCG(t/a)T(a/t)AATT) and, alone or fused to the protein oligomerization domain of phage lambda CI repressor, inhibits transcription from the hns promoter in vitro and in vivo. The importance of H-NS oligomerization is indicated by the fact that with an extended hns promoter construct (400 bp), which allows protein oligomerization, DNA binding and transcriptional repression are highly and almost equally efficient with native H-NS and H-NSctd::lambdaCI and much less effective with the monomeric H-NSctd. With a shorter (110 bp) construct, which does not sustain extensive protein oligomerization, transcriptional repression is less effective, but native H-NS, H-NSctd::lambdaCI, and monomeric H-NSctd have comparable activity on this construct. The specific H-NS-DNA interaction was investigated by NMR spectroscopy using monomeric H-NSctd and short DNA duplexes encompassing the H-NS target sequence of hns (TCCTTACATT) with the best fit (8 of 10 residues) to the H-NS-binding motif. H-NSctd binds specifically and with high affinity to the chosen duplexes via an overall electropositive surface involving four residues (Thr(109), Arg(113), Thr(114), and Ala(116)) belonging to the same protein loop and Glu(101). The DNA target is recognized by virtue of its sequence and of a TpA step that confers a structural irregularity to the B-DNA duplex.
- Subjects :
- Magnetic Resonance Spectroscopy
Amino Acid Motifs
Repressor
Biology
Biochemistry
DNA-binding protein
chemistry.chemical_compound
Bacterial Proteins
Transcription (biology)
Escherichia coli
Protein oligomerization
Nucleoid
Transcription, Chromatin, and Epigenetics
Settore BIO/10
Cloning, Molecular
Binding site
Promoter Regions, Genetic
Molecular Biology
Binding Sites
Base Sequence
C-terminus
DNA
Cell Biology
Molecular biology
DNA-Binding Proteins
Repressor Proteins
chemistry
Protein Multimerization
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 284
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....13404aba24c123d73cf5638c3fb84e2f