Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme

The native structure of the Azoarcus group I ri-bozyme is stabilized by the cooperative formation of tertiary interactions between double helical do-mains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme ac-tivity in physiological Mg2+ 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 scatter-ing, 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 Mg2+ concentrations and stabilized the active struc-ture 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 land-scape by stabilizing compact RNA structures without improving the specificity of self-assembly