期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 107, 期 26, 页码 11751-11756出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1004255107
关键词
5 '-phosphorothiolate; RNA catalysis; nucleolytic ribozymes; catalytic mechanism
资金
- National Institutes of Health [1R56AI081987-01]
- Cancer Research UK
- Howard Hughes Medical Institute
- Biotechnology and Biological Sciences Research Council [B17092] Funding Source: researchfish
Existing evidence suggests that the Varkud satellite (VS) ribozyme accelerates the cleavage of a specific phosphodiester bond using general acid-base catalysis. The key functionalities are the nucleobases of adenine 756 in helix VI of the ribozyme, and guanine 638 in the substrate stem loop. This results in a bell-shaped dependence of reaction rate on pH, corresponding to groups with pK(a) = 5.2 and 8.4. However, it is not possible from those data to determine which nucleobase is the acid, and which the base. We have therefore made substrates in which the 5' oxygen of the scissile phosphate is replaced by sulfur. This labilizes the leaving group, removing the requirement for general acid catalysis. This substitution restores full activity to the highly impaired A756G ribozyme, consistent with general acid catalysis by A756 in the unmodified ribozyme. The pH dependence of the cleavage of the phosphorothiolate-modified substrates is consistent with general base catalysis by nucleobase at position 638. We conclude that cleavage of the substrate by the VS ribozyme is catalyzed by deprotonation of the 2'-O nucleophile by G638 and protonation of the 5'-O leaving group by A756.
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