Lee Hui-Ting, Kilburn Duncan, Behrouzi Reza, Briber Robert M, Woodson Sarah A
Thomas C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA.
Thomas C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA Center for Neutron Scattering Research, National Institute of Standards and Technology, 100 Bureau Dr., Gaithersburg, MD 20899, USA.
Nucleic Acids Res. 2015 Jan;43(2):1170-6. doi: 10.1093/nar/gku1335. Epub 2014 Dec 24.
The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg(2+) concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg(2+) concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.
偶氮螺菌属I型核酶的天然结构通过双螺旋结构域之间三级相互作用的协同形成得以稳定。因此，即使是破坏这种三级相互作用网络的单个突变，也会在生理镁离子浓度下降低核酶活性。在此，我们报道与细胞内情况相当的分子拥挤效应能够补偿偶氮螺菌核酶中的去稳定化突变。利用小角X射线散射、天然聚丙烯酰胺凝胶电泳和活性测定法，比较了含有18%聚乙二醇1000（PEG1000）的稀溶液和拥挤溶液中的折叠自由能。拥挤剂分子使野生型和突变型核酶在相似的低镁离子浓度下折叠，并在生理条件下稳定了突变型核酶的活性结构。这种补偿作用有助于解释为什么核酶突变在细胞内通常比在试管中危害小。然而，拥挤效应并不能挽救由双突变体和三突变体形成的高比例折叠但活性较低的结构。我们得出结论，拥挤效应通过稳定紧密的RNA结构拓宽了适应性景观，但并未提高自组装的特异性。
