Kukolj G, Tolias P P, Autexier C, DuBow M S
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
EMBO J. 1989 Oct;8(10):3141-8. doi: 10.1002/j.1460-2075.1989.tb08467.x.
We have purified the 8.6 kd ner gene product (a lambda Cro-like protein which negatively regulates transcription from two divergent and overlapping promoters) from the Mu-like transposable bacteriophage D108. Chemical and enzymatic protection experiments show the D108 ner-operator to contain two perfect 11 bp (5'-CCG-TGAGCTAC-3') inverted repeats separated by an 8 bp AT-rich region. Ner makes base-specific contacts in the major groove spanning the 11 bp repeats and also interacts with regions flanking these sites such that its operator comprises five turns of the DNA helix. Furthermore, gel filtration chromatography and dimethyl suberimidate crosslinking experiments indicate that D108 Ner (at concentrations exceeding 5 microM) is a monomer in solution, yet crosslinks as a dimer when bound to its operator site. As a small (73 amino acids) monomeric protein, Ner does not display strong homology with any known DNA-binding proteins. By virtue of the interactions with its operator it appears to bind DNA in a markedly different manner from other known prokaryotic repressors thus adding to the growing catalog of protein motifs used for specific binding to DNA.
我们从类Mu转座噬菌体D108中纯化出了8.6kd的ner基因产物（一种类似λCro的蛋白，对两个反向且重叠的启动子的转录起负调控作用）。化学和酶促保护实验表明，D108的ner操纵基因含有两个完美的11bp（5'-CCG-TGAGCTAC-3'）反向重复序列，中间由一个8bp富含AT的区域隔开。Ner在跨越11bp重复序列的大沟中与碱基发生特异性接触，并且还与这些位点两侧的区域相互作用，使其操纵基因包含五圈DNA螺旋。此外，凝胶过滤色谱法和亚胺基二琥珀酸二甲酯交联实验表明，D108 Ner（浓度超过5μM时）在溶液中是单体，但与操纵基因位点结合时会交联形成二聚体。作为一种小的（73个氨基酸）单体蛋白，Ner与任何已知的DNA结合蛋白都没有很强的同源性。由于与操纵基因的相互作用，它似乎以一种与其他已知原核生物阻遏物明显不同的方式结合DNA，从而增加了用于与DNA特异性结合的蛋白质基序的种类。
