Identification of mutations necessary for full growth levels in the evolved spliceozyme W11. (A) Growth of E. coli cells expressing different spliceozyme variants on plates containing 100 μg/mL of chloramphenicol. In addition to the parent ribozyme and the evolved ribozyme W11, variants of W11 with reversions of indicated mutations were analyzed. The reverted mutations are given below the graph, with AA5′CU describing the mutation of two 5′-terminal nucleotides to CU, and A3′C describing the mutation of the 3′-terminal nucleotide to C. The OD600 was measured from growth on plates, and was normalized to the OD600 from plate cultures containing ampicillin (i.e., viable cells containing plasmid). Error bars are standard deviations from biological triplicates. (B) Secondary structure of the evolved spliceozyme W11, with evolved mutations indicated in green and with green circles. New base pairs created by the mutation are highlighted in green, and base pairs deleted by the mutation are highlighted in red.
