Figure 4.

Chemical and enzymatic probing of the bottom of the P2 stem. (A) Autoradiogram of a 10% PAGE gel of in-line probing performed on 5′-end-labelled wild-type and mutated trans-acting ribozymes. Both alkaline and RNase T1 hydrolyses of the wild-type ribozyme were performed in order to determine the location of each position (lanes OH and T1, respectively). In-line probing of the wild-type ribozyme (1, 2), the RzC₂₄U,C₂₅U,G₄₀U,G₄₁U (3, 4) and the RzA₇₈U,A₇₉U (5, 6) mutants are shown. The experiments were performed either in the absence (−) or the presence (+) of the SdA4 analogue. The secondary structure motifs are identified on the left. (B) Autoradiogram of a 10% PAGE gel of RNase V1 probing performed on 5′-end-labelled wild-type and mutated cis-acting ribozymes. Both alkaline and RNase T1 hydrolyses of the wild-type sequence were performed in order to determine the location of each position (lanes OH and T1, respectively). Lanes 1 to 4 correspond to the wild-type sequence (C₁₉G₈₁G₈₀) and the mutants RzC₁₉,G₈₁A,G₈₀A, RzC₁₉,G₈₁AG₈₀ and RzC₁₉G₈₁G₈₀A, respectively. The positions of the C₁₉ and C₂₄ (used to establish the relative level of hydrolysis) are indicated on the right. (C) Histogram of the relative levels of RNase V1 hydrolysis of C₁₉ for each ribozyme.