Nucleic Acids Research, 1985, Vol. 13, No. 21 7759-7779
© 1985
Articles |
Self-catalyzed cyclization of the intervening sequence RNA of Tetrahymena: inhibition by methidiumpropyl-EDTA and localization of the major dye binding sites
Department of Chemistry, University of Colorado Boulder, CO 80309, USA
Received May 27, 1985. Revised October 4, 1985. Accepted October 4, 1985.
The intervening sequence (IVS) excised from the rRNA precursor of Tetrahymens thermophils is converted to a covalently closed circular RNA in the absence of proteins in vitro. This self-catalyzed cyclization reaction is inhibited by the intercalating dye methidiumpropyl · EDTA (MPE; R.P. Hertzberg and P.B. Dervan (1982) J. Am. Chem. Soc. 104, 313–315). The MPE binding sites have been localized by mapping the sites of MPE · Fe(II) cleavage of the IVS RNA. There are three major binding sites within the 414 nucleotide IVS RNA. Two of these sites coincide with the A·B and 9L·2 pairings. These are structural elements that are conserved in all group I introns and are implicated as being functionally important for splicing. We propose that interaction of MPE with these sites is responsible for dye inhibition of cyclization. The reactions of MPE·Fe(II) with an RNA of known structure, tRNAphe, and with the IVS RNA were studied as a function of temperature, ionic strength and ethidium concentration. Based on the comparison of the reaction with these two RNAs, we conclude that the dye is a very useful probe for structural regions of large RNAs, while it provides more limited structural information about the small, compact tRNA molecule.