Sequence-specific Recognition of DNA by the C-terminal Domain of Nucleoid-associated Protein H-NS*
Sequence-specific Recognition of DNA by the C-terminal Domain of Nucleoid-associated Protein H-NS*
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核相关蛋白 H-NS* C 末端结构域对 DNA 的序列特异性识别
DOI：
10.1074/jbc.m109.044313
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
C. Gualerzi
中科院分区：
文献类型：
--
作者：
M. Sette;R. Spurio;E. Trotta;Cinzia Brandizi;A. Brandi;C. Pon;G. Barbato;R. Boelens;C. Gualerzi
核相关蛋白H-NS的c端结构域（H-NSctd）包含识别其特定DNA靶标所必需和充分的分子决定因子。H-NSctd保护了H-NS敏感的hns启动子的一些短DNA片段，这些片段的序列与最近发现的H-NS一致基序(tCG（t/a) t (a/t)AATT）密切匹配，并且单独或融合到噬菌体λ CI抑制因子的蛋白寡聚化结构域，在体外和体内抑制hns启动子的转录。H-NS寡聚化的重要性体现在这样一个事实上，即具有扩展的hns启动子结构（400 bp），允许蛋白质寡聚化，DNA结合和转录抑制与天然H-NS和H-NSctd::λCI的效率几乎相同，而与单体H-NSctd的效率要低得多。对于较短的（110 bp）结构，它不会维持广泛的蛋白质寡聚化，转录抑制效果较差，但天然H-NS， H-NSctd::λCI和单体H-NSctd对该结构具有相当的活性。利用单体H-NSctd和包含H-NS靶序列（TCCTTACATT）的短DNA双链（10个残基中的8个）与H-NS结合基序最匹配)，通过核磁共振光谱研究了H-NS与DNA的特异性相互作用。H-NSctd通过一个包含四个残基（Thr109, Arg113， Thr114和Ala116）的整体电正表面与所选的双链结合，具有特异性和高亲和力，这些残基属于同一蛋白环和Glu101。DNA靶标通过其序列和赋予B-DNA双链结构不规则性的TpA步骤被识别。
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 λ 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::λCI 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::λCI, 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 (Thr109, Arg113, Thr114, and Ala116) belonging to the same protein loop and Glu101. 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.
影响因子：
5.6
作者：
Protozanova, E;Yakovchuk, P;Frank-Kamenetskii, MD
通讯作者：
Frank-Kamenetskii, MD