Identification of phosphate groups important to self-splicing of theTetrahymenarRNA intron as determined by phosphorothioate substitution

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Abstract
The group I intron from the rRNA precursor of Tetrahymena undergoes self-splicing. The intron RNA catalyst contains about 400 phosphate groups. Their role in catalysis has been investigated using phosphorothioate substituted RNA. In such RNA one of the peripheral oxygens of the phosphodiesters is replaced with sulfur. Incorporation of adenosine 5'' phosphorothioate in either the 5'' or 3'' half of the ribozyme blocked splicing whereas incorporation of uridine 5'' phosphorothioate only blocked splicing if the substitution was in the 3'' half of the molecule. Modification-interference assays located two major and three minor inhibitory phosphorothioate substitutions suggesting that the corresponding phosphate plays a significant role in self-splicing. These are all located in the most highly conserved region of the intron.