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.
Highlights
Nucleoid-associated DNA-binding protein H-NS plays the dual role of architectural protein of the nucleoid and regulator of expression of a large number of genes
H-NS binds preferentially to DNA bends (4, 10 –13) endowed with special geometric features (14, 15), but the quantitative aspects of this preference remained a matter of dispute, because the very extended, ill-defined, and diffuse footprinting patterns often produced by H-NS on its target promoters (16, 17) suggested that H-NS was devoid of any DNA binding specificity (18)
Instead of using chimerae having the DNA binding capacity of a lambdoid repressor and the protein oligomerization property of H-NS, we have constructed a chimera in which the DNA binding property is provided by H-NSctd (H-NS(⌬1– 88)) and the protein oligomerization function is provided by the C-domain of bacteriophage CI repressor (CI(⌬1–92)) (Fig. 1A)
Summary
Nucleoid-associated DNA-binding protein H-NS plays the dual role of architectural protein of the nucleoid and regulator of expression of a large number of genes. It was suggested that the consensus sequences represent the specific target where H-NS nucleation initially starts before subsequent protein oligomerization leads to the assembly of the transcriptional repression complex. Following initial binding to nucleation sites strategically located in a given promoter, H-NS-H-NS interactions could promote bridging between the two sides of a DNA bend (17, 18, 20 –22) and/or the lateral condensation of distant stretches of duplex DNA (5), two processes considered to be key features by which H-NS exercises its roles of transcriptional repressor and architectural organizer of the nucleoid (19). The protein oligomerization functions of H-NS is generally attributed to the N-terminal domain (H-NSntd), whereas the C-domain (H-NSctd) is considered responsible for DNA binding Important roles in protein oligomerization and DNA binding have been attributed to H-NSctd and to H-NSntd, respectively (24 –26)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
