Smith T L, Sauer R T
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
J Mol Biol. 1995 Jun 16;249(4):729-42. doi: 10.1006/jmbi.1995.0332.
Dimers of P22 Arc repressor bind to half-sites of the 21 bp arc operator and interact cooperatively to stabilize a DNA-bound tetramer. Mutation of Ser35 (a residue in the dimer-dimer interface) to Arg or Leu disrupts cooperative binding. The mutant proteins have near wild-type stabilities, give operator footprints like wild-type, and prevent binding of RNA polymerase to the Pant promoter in vitro. These mutants are, however, largely inactive in vivo. Thus, although cooperativity is not structurally required for repression, it appears that the additional DNA-binding energy from dimer-dimer cooperativity is required for normal biological function. Altering the spacing between the DNA half-sites by even one base-pair eliminates dimer-dimer cooperativity, indicating that Arc dimers need to be oriented correctly by half-site binding to allow the interactions that stabilize the tetrameric complex.
P22 Arc阻遏物的二聚体与21 bp的arc操纵基因的半位点结合，并协同相互作用以稳定结合于DNA的四聚体。将Ser35（二聚体 - 二聚体界面中的一个残基）突变为精氨酸或亮氨酸会破坏协同结合。突变蛋白具有接近野生型的稳定性，产生与野生型相似的操纵基因足迹，并在体外阻止RNA聚合酶与Pant启动子结合。然而，这些突变体在体内基本上是无活性的。因此，虽然协同作用在结构上对于阻遏不是必需的，但似乎二聚体 - 二聚体协同作用产生的额外DNA结合能量对于正常生物学功能是必需的。将DNA半位点之间的间距改变哪怕一个碱基对都会消除二聚体 - 二聚体协同作用，这表明Arc二聚体需要通过半位点结合正确定向，以允许稳定四聚体复合物的相互作用。
