Journal
PLOS BIOLOGY
Volume 3, Issue 9, Pages 1536-1548Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pbio.0030277
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Funding
- NIGMS NIH HHS [GM49243, R37 GM049243, R01 GM049243, T32 GM008720, 2 T32 GM008720-06] Funding Source: Medline
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The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate( s). In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (M-A, M-B, and M-C) that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-S-P phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for M-C. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.
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