Talbot S J, Goodman S, Bates S R, Fishwick C W, Stockley P G
Department of Genetics, School of Chemistry, University of Leeds, UK.
Nucleic Acids Res. 1990 Jun 25;18(12):3521-8. doi: 10.1093/nar/18.12.3521.
Synthetic oligoribonucleotides have been used to probe the interaction of MS2 coat protein with the translational operator of the MS2 replicase gene. We have investigated the possible formation of a transient covalent bond between the single-stranded uridine residue, at position -5, and a cysteine side-chain on the coat protein, by the incorporation of a chemically modified residue (5-BrU) at this position. This chemically synthesised operator variant has a binding constant of between 10 and 50 times greater than that of the wild type and is therefore comparable with the tight binding variant having a cytidine substituted at the -5 position. Dissociation kinetics show that the complex with the 5-BrU operator is more stable than the -5C variant; a result which is consistent with the formation of a Michael adduct at the -5 position. In addition, a number of other chemical variants of the operator have been analysed. These include operators incorporating deoxyadenine residues at each of the important single-stranded adenine sites. Recently the Michael adduct proposal has been challenged on the basis of mutagenesis of the coat protein cysteine residues. These results are discussed in the light of our data in support of Michael adduct formation.
合成寡核糖核苷酸已被用于探究MS2外壳蛋白与MS2复制酶基因翻译操纵子之间的相互作用。我们通过在-5位掺入化学修饰的残基（5-溴尿苷，5-BrU），研究了-5位的单链尿苷残基与外壳蛋白上的半胱氨酸侧链之间可能形成的瞬时共价键。这种化学合成的操纵子变体的结合常数比野生型大10至50倍，因此与在-5位用胞苷取代的紧密结合变体相当。解离动力学表明，与5-BrU操纵子形成的复合物比-5C变体更稳定；这一结果与在-5位形成迈克尔加成物一致。此外，还分析了操纵子的一些其他化学变体。这些包括在每个重要的单链腺嘌呤位点掺入脱氧腺嘌呤残基的操纵子。最近，基于外壳蛋白半胱氨酸残基的诱变，迈克尔加成物的提议受到了挑战。根据我们支持迈克尔加成物形成的数据对这些结果进行了讨论。
