The mechanism by which group II introns cleave the correct phosphodiester linkage was investigated by studying the reaction of mutant substrates with a ribozyme derived from intron ai5gamma. While fidelity was found to be quite high in most cases, a single mutation on the substrate (+1C) resulted in a dramatic loss of fidelity. When this mutation was combined with a second mutation that induces a bulge in the exon binding site 1/intron binding site 1 (EBS1/IBS1) duplex, the base-pairing register of the EBS1/IBS1 duplex was shifted and the cleavage site moved to a downstream position on the substrate. Conversely, when mismatches were incorporated at the EBS1/IBS1 terminus, the duplex was effectively truncated and cleavage occurred at an upstream site. Taken together, these data demonstrate that the cleavage site of a group II intron ribozyme can be tuned at will by manipulating the thermodynamic stability and structure of the EBS1/IBS1 pairing. The results are consistent with a model in which the cleavage site is not designated through recognition of specific nucleotides (such as the 5'-terminal residue of EBS1). Instead, the ribozyme detects a structure at the junction between single and double-stranded residues on the bound substrate. This finding explains the puzzling lack of phylogenetic conservation in ribozyme and substrate sequences near group II intron target sites.
Copyright 2001 Academic Press.