Lim F, Downey T P, Peabody D S
Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA.
J Biol Chem. 2001 Jun 22;276(25):22507-13. doi: 10.1074/jbc.M102411200. Epub 2001 Apr 16.
PP7 is a single-strand RNA bacteriophage of Pseudomonas aeroginosa and a distant relative to coliphages like MS2 and Qbeta. Here we show that PP7 coat protein is a specific RNA-binding protein, capable of repressing the translation of sequences fused to the translation initiation region of PP7 replicase. Its RNA binding activity is specific since it represses the translational operator of PP7, but does not repress the operators of the MS2 or Qbeta phages. Conditions for the purification of coat protein and for the reconstitution of its RNA binding activity from disaggregated virus-like particles were established. Its dissociation constant for PP7 operator RNA in vitro was determined to be about 1 nm. Using a genetic system in which coat protein represses translation of a replicase-beta-galactosidase fusion protein, amino acid residues important for binding of PP7 RNA were identified.
PP7是铜绿假单胞菌的一种单链RNA噬菌体，与MS2和Qβ等大肠杆菌噬菌体是远亲。我们在此表明，PP7外壳蛋白是一种特异性RNA结合蛋白，能够抑制与PP7复制酶翻译起始区域融合的序列的翻译。其RNA结合活性具有特异性，因为它能抑制PP7的翻译操纵子，但不抑制MS2或Qβ噬菌体的操纵子。建立了外壳蛋白的纯化条件以及从解聚的病毒样颗粒中重建其RNA结合活性的条件。测定其在体外对PP7操纵子RNA的解离常数约为1纳米。利用一个外壳蛋白抑制复制酶 - β - 半乳糖苷酶融合蛋白翻译的遗传系统，鉴定了对PP7 RNA结合重要的氨基酸残基。
