Complementation of RNA binding site mutations in MS2 coat protein heterodimers
Complementation of RNA binding site mutations in MS2 coat protein heterodimers
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DOI：
10.1093/nar/24.12.2352
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发表时间：
1996-06-15
影响因子：
14.9
通讯作者：
Lim, F
中科院分区：
文献类型：
--
作者：
Peabody, DS;Lim, F
噬菌体MS 2的外壳蛋白作为对称二聚体结合不对称RNA发夹发挥功能。这意味着存在两个等价的RNA结合位点，它们通过2重对称轴相互关联。在这个观点中，由赋予阻遏物缺陷表型的突变所定义的对称结合位点是这两个不对称位点的合成图。为了确定RNA配体是否与二聚体的两个亚基上的氨基酸残基相互作用，并希望构建RNA结合位点的功能图，我们进行了异源二聚体互补实验。利用它们的N-和C-末端的物理接近性，二聚体的两个亚基被遗传融合，产生复制的外壳蛋白，其正常折叠，并允许构建含有阻遏物缺陷突变的所有可能的成对组合的强制性异二聚体的功能等价物。某些异源二聚体中阻遏物功能的恢复表明，单个RNA分子与二聚体的两个亚基相互作用，并允许构建结合位点的功能图。
The coat protein of bacteriophage MS2 functions as a symmetric dimer to bind an asymmetric RNA hairpin. This implies the existence of two equivalent RNA binding sites related to one another by a 2-fold symmetry axis. In this view the symmetric binding site defined by mutations conferring the repressor-defective phenotype is a composite picture of these two asymmetric sites. In order to determine whether the RNA ligand interacts with amino acid residues on both subunits of the dimer and in the hope of constructing a funtional map of the RNA binding site, we performed heterodimer complementation experiments. Taking advantage of the physical proximity of their N- and C-termini, the two subunits of the dimer were genetically fused, producing a duplicated coat protein which folds normally and allows the construction of the functional equivalent of obligatory heterodimers containing all possible pairwise combinations of the repressor-defective mutations. The restoration of repressor function in certain heterodimers shows that a single RNA molecule interacts with both subunits of the dimer and allows the construction of a functional map of the binding site.