Effect of the 3′-terminal intron sequence on spliceozyme activity in E. coli cells.

(A) Secondary structure of the spliceozyme (black), with the substrate exons in red, and the position of the randomized N10 sequence in the 3′-terminal region of the intron (blue). The helices P9.0 and P9.2 are labeled. (B) List of 20 analyzed clones, sorted according to the resulting activity in cells. The clone name is given on the left, the growth on LB-agar plates containing chloramphenicol is shown as horizontal columns, and the N10 sequence is given on the right. Clone names containing ‘L’ indicate the unselected library, while ‘S’ indicates a selection step with chloramphenicol. The letter ‘j’ denotes that the randomized region is located near the 3′-terminus of the CAT mRNA intron. The growth was measured on plates containing 10 µg/mL chloramphenicol and normalized to growth on medium without chloramphenicol. The sequence at the top (WT) corresponds to the wild type sequence of the Tetrahymena ribozyme. Nucleotides that are identical with the wild type sequence are colored in blue. The position 10 tolerates a U, which is consistent with its base-pairing role, and is colored in red. Errors are standard deviations from three experiments.