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Nucleic Acids Research, 1989, Vol. 17, No. 24 10281-10293
© 1989

Articles

Identification of phosphate groups important to self-splicing of the Tetrahymena rRNA intron as determined by phosphorothioate substitution

Richard B. Waring

Department of Biology, Temple University Philadelphia, PA 19122, USA

Received September 22, 1989. Revised November 13, 1989. Accepted November 13, 1989.

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 phosphates play a significant role in self-splicing. These are all located in the most highly conserved region of the intron.


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