Enzymatic analysis of the secondary structure of Apt1. (A) The secondary structure of Apt1 predicted by RNAstructure 4.4. (Boldface) The 5′- and 3′-constant sequences of Apt1 for primer annealing; (red) the consensus sequences. The cleavage points of RNase V1 (open arrowhead), RNase T1 (solid arrowhead), and mung bean nuclease (striped arrowhead) and points uncleaved by RNase T1 (gray arrowhead) are indicated on the secondary structure of Apt1. The arrowhead length indicates the signal intensity of the digested fragments. (Dotted line) A predicted base pair. The signals >A55 were too strong to identify individual signals (lane 6 of panel B); therefore, the cleavage points by mung bean nuclease are not indicated from C56 to the 3′ terminus. (B) Autoradiogram of the RNase probing pattern of Apt1. The 5′-[32P]-labeled Apt1 (lane 2) was partially digested with alkali (lane 1), RNase T1 in the presence of 7 M urea (lane 3), RNase T1 (lane 4), RNase V1 (lane 5), and mung bean nuclease (lane 6). Digests were separated using denaturing polyacrylamide gel electrophoresis. (Left side of the autoradiogram) The positions of G-residues. The bar indicates the position of the full-length Apt1 (63-mer). (Right side) The estimated bases digested by RNase V1 and mung bean nuclease.
