To examine hammerhead ribozyme structure and vulnerability to cellular nucleases, ribozymes were incubated with soluble extracts from Escherichia coli, and cleavage sites were identified by primer extension analysis. Mapping of endonuclease-sensitive sites revealed that the most sensitive were in the 3'-substrate-binding region of the ribozyme. The catalytic domain was much less susceptible, although some cleavage preference was seen at two positions known to be twisted out of parallel stacking in a ribozyme-substrate analogue complex. Changes in substrate-binding domain nucleotide sequence had no effect on cleavage patterns of catalytic domains. Hammerhead ribozymes, in solution and free from substrate, appear to have structurally independent, asymmetrically arranged domains.
All Science Journal Classification (ASJC) codes
- Escherichia coli extracts
- RNA structure
- Ribonuclease I
- rna mutation