Witherell G W, Wu H N, Uhlenbeck O C
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Biochemistry. 1990 Dec 18;29(50):11051-7. doi: 10.1021/bi00502a006.
The binding of the R17 coat protein to synthetic RNAs containing one or two coat protein binding sites was characterized by using nitrocellulose filter and gel-retention assays. RNAs with two available sites bound coat protein in a cooperative manner, resulting in a higher affinity and reduced sensitivity to pH, ionic strength, and temperature when compared with RNAs containing only a single site. The cooperativity can contribute up to -5 kcal/mol to the overall binding affinity with the greatest cooperativity found at low pH, high ionic strength, and high temperatures. Similar solution properties for the encapsidation of the related fr and f2 phage suggest that the cooperativity is due to favorable interactions between the two coat proteins bound to the RNA. This system therefore resembles an intermediate state of phage assembly. No cooperative binding was observed for RNAs containing a single site and a 5' or 3' extension of nonspecific sequence, indicating that R17 coat protein has a very low nonspecific binding affinity. Unexpectedly weak binding was observed for several RNAs due to the presence of alternative conformational states of the RNA.
通过使用硝酸纤维素滤膜和凝胶滞留分析，对R17外壳蛋白与含有一个或两个外壳蛋白结合位点的合成RNA的结合进行了表征。具有两个可用位点的RNA以协同方式结合外壳蛋白，与仅含有单个位点的RNA相比，具有更高的亲和力，并且对pH、离子强度和温度的敏感性降低。这种协同作用对整体结合亲和力的贡献可达-5千卡/摩尔，在低pH、高离子强度和高温下协同作用最大。相关的fr和f2噬菌体衣壳化的类似溶液性质表明，协同作用是由于与RNA结合的两个外壳蛋白之间的有利相互作用。因此，该系统类似于噬菌体组装的中间状态。对于含有单个位点和5'或3'非特异性序列延伸的RNA，未观察到协同结合，这表明R17外壳蛋白具有非常低的非特异性结合亲和力。由于RNA存在替代构象状态，观察到几种RNA的结合出乎意料地弱。
