Spingola M, Peabody D S
Department of Cell Biology, University of New Mexico School of Medicine and Cancer Research and Treatment Center, Albuquerque, NM 87131, USA.
Nucleic Acids Res. 1997 Jul 15;25(14):2808-15. doi: 10.1093/nar/25.14.2808.
The coat proteins of the RNA phages MS2 and Qbetaare structurally homologous, yet they specifically bind different RNA structures. In an effort to identify the basis of RNA binding specificity we sought to isolate mutants that convert MS2 coat protein to the RNA binding specificity of Qbeta. A library of mutations was created which selectively substitutes amino acids within the RNA binding site. Genetic selection for the ability to repress translation from the Qbetatranslational operator led to the isolation of several MS2 mutants that acquired binding activity for QbetaRNA. Some of these also had reduced abilities to repress translation from the MS2 translational operator. These changes in RNA binding specificity were the results of substitutions of amino acid residues 87 and 89. Additional codon- directed mutagenesis experiments confirmed earlier results showing that the identity of Asn87 is important for specific binding of MS2 RNA. Glu89, on the other hand, is not required for recognition of MS2 RNA, but prevents binding of QbetaRNA.
RNA噬菌体MS2和Qβ的外壳蛋白在结构上具有同源性，但它们特异性结合不同的RNA结构。为了确定RNA结合特异性的基础，我们试图分离出能将MS2外壳蛋白转化为具有QβRNA结合特异性的突变体。构建了一个突变文库，该文库选择性地替换RNA结合位点内的氨基酸。通过对抑制Qβ翻译起始区翻译能力的遗传筛选，分离出了几个对QβRNA具有结合活性的MS2突变体。其中一些突变体抑制MS2翻译起始区翻译的能力也有所降低。RNA结合特异性的这些变化是由于氨基酸残基87和89的替换所致。额外的定点诱变实验证实了早期的结果，表明Asn87的身份对于MS2 RNA的特异性结合很重要。另一方面，Glu89对于识别MS2 RNA不是必需的，但会阻止QβRNA的结合。
