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.
Su
L J
LJ
Department of Biochemistry and Molecular Biophysics and the Howard Hughes Medical Institute, Columbia University, 630 W. 168th Street, New York, NY, 10032, USA.
Qin
P Z
PZ
Michels
W J
WJ
Pyle
A M
AM
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