In vitro selection of second site revertants analysis of the hairpin ribozyme active site.
We have used in vitro genetics to evaluate the function and interactions of the conserved base G8 in the hairpin ribozyme catalytic RNA. Second site revertant selection for a G8X mutant, where X is any of the other three natural nucleobases, yielded a family of second site suppressors of the G8U mutant, but not of G8C or G8A, indicating that only G and U can be tolerated at position 8 of the ribozyme. This result is consistent with recent observations that point to the functional importance of G8 N-1 in the chemistry of catalysis by this ribozyme reaction. Suppression of the G8U mutation was observed when changes were made directly across loop A from the mutated base at substrate position +2 or positions +2 and +3 in combination. The same changes made in the context of the natural G8 sequence resulted in a very large drop in activity. Thus, the G8U mutation results in a change in specificity of the ribozyme from 5'-N / GUC-3' to 5'-N / GCU-3'. The results presented imply that G8 interacts directly with U+2 during catalysis. We propose that this interaction favors the correct positioning of the catalytic determinants of G8. The implications for the folding of the ribozyme and the catalytic mechanism are discussed.
Sargueil
Bruno
B
Centre de Génétique Moléculaire, CNRS, 91190 Gif-sur-Yvette, France. sargueil@cgm.cnrs-gif.fr
Hampel
Ken J
KJ
Lambert
Dominic
D
Burke
John M
JM
D001483:Base Sequence; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D002384:Catalysis; D004247:DNA / Q000737:chemistry; D005821:Genetic Techniques; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry*; D013997:Time Factors
D009841:Oligonucleotides; D016337:RNA, Catalytic; C418481:hairpin ribozyme; D012313:RNA; D004247:DNA