Figure 2.

Cleavage activity in the cis configuration. A cis-acting snorbozyme was constructed in which a hammerhead sequence (52 nt) was inserted into the full-size U3* molecule in place of nucleotides 10–67. The Northern blot identifies RNAs expressed in yeast (in vivo) or with T7 RNA polymerase in vitro (in vitro 1). To verify that cleavage and subsequent trimming of the in vivo-expressed snorbozyme did not occur during RNA isolation, the in vitro-transcribed molecules were added to intact yeast cells and passed through the same RNA isolation procedure (in vitro 2). + refers to the active ribozyme; − refers to the defective ribozyme (see Fig. 1B). Additional control samples correspond to total RNA isolated from cells expressing the U3* or empty host vector (V). The biochemical events leading to the final products obtained in vivo and in vitro are shown schematically. The P2 product is not identified in the Northern blot (in vitro lanes) because of its small size (14 nt).