Johansson H E, Dertinger D, LeCuyer K A, Behlen L S, Greef C H, Uhlenbeck O C
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA.
Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9244-9. doi: 10.1073/pnas.95.16.9244.
Most mutations in the sequence of the RNA hairpin that specifically binds MS2 coat protein either reduce the binding affinity or have no effect. However, one RNA mutation, a uracil to cytosine change in the loop, has the unusual property of increasing the binding affinity to the protein by nearly 100-fold. Guided by the structure of the protein-RNA complex, we used a series of protein mutations and RNA modifications to evaluate the thermodynamic basis for the improved affinity: The tight binding of the cytosine mutation is due to (i) the amino group of the cytosine residue making an intra-RNA hydrogen bond that increases the propensity of the free RNA to adopt the structure seen in the complex and (ii) the increased affinity of hydrogen bonds between the protein and a phosphate two bases away from the cytosine residue. The data are in good agreement with a recent comparison of the cocrystal structures of the two complexes, where small differences in the two structures are seen at the thermodynamically important sites.
大多数特异性结合MS2外壳蛋白的RNA发夹序列中的突变要么降低结合亲和力，要么没有影响。然而，一种RNA突变，即环中尿嘧啶到胞嘧啶的变化，具有不同寻常的特性，可使与该蛋白的结合亲和力提高近100倍。在蛋白质-RNA复合物结构的指导下，我们使用了一系列蛋白质突变和RNA修饰来评估亲和力提高的热力学基础：胞嘧啶突变的紧密结合归因于（i）胞嘧啶残基的氨基形成了一个RNA内氢键，增加了游离RNA采用复合物中所见结构的倾向，以及（ii）蛋白质与胞嘧啶残基下游两个碱基处的磷酸基团之间氢键亲和力的增加。这些数据与最近对两种复合物共晶体结构的比较非常吻合，在热力学重要位点上可以看到两种结构存在微小差异。
