Dertinger D, Dale T, Uhlenbeck O C
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA.
J Mol Biol. 2001 Dec 7;314(4):649-54. doi: 10.1006/jmbi.2001.5132.
The interaction between the MS2 bacteriophage coat protein homodimer and its cognate RNA hairpin is facilitated by 21 different RNA-protein contacts. In one of these contacts, the 2'-hydroxyl group at ribose -5 of the RNA acts as a hydrogen bond donor to Glu63 in one subunit of the protein. Previous experiments showed that substitution of ribose -5 with deoxyribose resulted in a 24-fold decrease in binding affinity between RNA and protein. Using a protein where the two MS2 monomers were fused to increase stability, the contribution of this contact to the overall binding affinity was investigated by site-directed mutagenesis. When Glu63 was substituted with glutamine, aspartate, or alanine, the binding affinity of the hairpin for the protein was weakened by 12 to 100-fold, similar to that observed with deoxyribose at position -5. However, the specificity of the three mutant proteins for RNAs with various modifications at the 2'-position of ribose -5 differed dramatically. While the Glu63Asp protein resembled the wild-type protein in preferring the 2'-hydroxyl group over a proton or a bulky 2'-substituent, both the Glu63Ala and Glu63Gln proteins preferred bulky 2'-substituents over the 2'-hydroxyl group by more than 100-fold. These experiments emphasize the ease with which the specificity of a protein-nucleic acid interaction can be changed at thermodynamically important sites.
MS2噬菌体外壳蛋白同型二聚体与其同源RNA发夹之间的相互作用由21种不同的RNA-蛋白质接触促进。在其中一种接触中，RNA核糖-5位的2'-羟基作为氢键供体与蛋白质一个亚基中的Glu63形成氢键。先前的实验表明，用脱氧核糖取代核糖-5会导致RNA与蛋白质之间的结合亲和力下降24倍。使用将两个MS2单体融合以提高稳定性的蛋白质，通过定点诱变研究了这种接触对整体结合亲和力的贡献。当Glu63被谷氨酰胺、天冬氨酸或丙氨酸取代时，发夹对蛋白质的结合亲和力减弱了12至100倍，类似于在-5位用脱氧核糖观察到的情况。然而，这三种突变蛋白对核糖-5的2'-位具有各种修饰的RNA的特异性差异很大。虽然Glu63Asp蛋白在优先选择2'-羟基而非质子或庞大的2'-取代基方面类似于野生型蛋白，但Glu63Ala和Glu63Gln蛋白在优先选择庞大的2'-取代基而非2'-羟基方面比野生型蛋白高出100倍以上。这些实验强调了在热力学重要位点改变蛋白质-核酸相互作用特异性的容易程度。
