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 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.
Department of Biochemistry and Molecular Biophysics and the Howard Hughes Medical Institute, Columbia University, 630 W. 168th Street, New York, NY, 10032, USA.
L J
LJ
Su
P Z
PZ
Qin
W J
WJ
Michels
A M
AM
Pyle
D020137:Base Pair Mismatch; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D005091:Exons / Q000235:genetics; D007438:Introns / Q000235:genetics*; D007700:Kinetics; D008957:Models, Genetic; D009154:Mutation / Q000235:genetics; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D002413:Cations, Divalent; D012322:RNA Precursors; D016337:RNA, Catalytic; D015719:Single-Strand Specific DNA and RNA Endonucleases