FIGURE 5.

Identification of mutations that increased ribozyme activity, among the 11 mutations in clone IV-12-10. A, activity of clone IV-12-10 variants that carried single reversion mutations toward the parent ribozyme. Activity was measured as growth on LB agar containing 200 μg/ml chloramphenicol. Note the logarithmic scale that shows the growth as measured as A₆₀₀. Growth of the parent ribozyme is shown as comparison, and a horizontal dashed line is shown for comparison with clone IV-12-10. The red arrow indicates a mutation reversion with >10-fold effect (G9U); the blue arrows indicate mutation reversions with <10-fold effect. B, five mutations were necessary to mediate full activity. A ribozyme containing only the four mutations identified in subfigure A (ribozyme variant M4) did not mediate full activity compared with the IV-12-10 variant. Three additional candidate mutations were added to ribozyme M4 and measured for growth in the presence of 200 μg/ml chloramphenicol. Mutation U241A, in combination with the four mutations identified in subfigure A, was found to be necessary and sufficient for full activity observed in the IV-12-10 variant. Secondary structure of the ribozyme with the positions of beneficial mutations indicated. Colorcoding is as in A. The helices containing these mutations are labeled as 5′-duplex and P6b.