Sequence-specific recognition of DNA by the C-terminal domain of nucleoid-associated protein H-NS.
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.
Sette
Marco
M
From the Department of Chemical Sciences and Technology, University of Rome-Tor Vergata, 00133 Rome, Italy.
Spurio
Roberto
R
Trotta
Edoardo
E
Brandizi
Cinzia
C
Brandi
Anna
A
Pon
Cynthia L
CL
Barbato
Gaetano
G
Boelens
Rolf
R
Gualerzi
Claudio O
CO
D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D011401:Promoter Regions, Genetic; D055503:Protein Multimerization; D012097:Repressor Proteins
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; D012097:Repressor Proteins; D004247:DNA