Guiding ribozyme cleavage through motif recognition: The mechanism of cleavage site selection by a group II intron ribozyme

The mechanism by which group II introns cleave the correct phosphodiester l inkage was investigated by studying the reaction of mutant substrates with a ribozyme derived from intron ai5 gamma. While fidelity was found to be qu ite high in most cases, a single mutation on the substrate (+1C) resulted i n a dramatic loss of fidelity. When this mutation was combined with a secon d mutation that induces a bulge in the exon binding site 1/intron binding s ite 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 su bstrate. Conversely, when mismatches were incorporated at the EBS1/IBS1 ter minus, the duplex was effectively truncated and cleavage occurred at an ups tream site. Taken together, these data demonstrate that the cleavage site o f a group II intron ribozyme can be tuned at will by manipulating the therm odynamic stability and structure of the EBS1/IBS1 pairing. The results are consistent with a model in which the cleavage site is not designated throug h recognition of specific nucleotides (such as the 5'-terminal residue of E BS1). Instead, the ribozyme detects a structure at the junction between sin gle and double-stranded residues on the bound substrate. This finding expla ins the puzzling lack of phylogenetic conservation in ribozyme and substrat e sequences near group II intron target sites. (C) 2001 Academic Press.