CLEAVAGE OF TRANSFER-RNA WITH IMIDAZOLE AND SPERMINE IMIDAZOLE CONSTRUCTS - A NEW APPROACH FOR PROBING RNA STRUCTURE

Hydrolysis of RNA in imidazole buffer and by spermine-imidazole conjug ates has been investigated. The RNA models were yeast tRNA(Asp) and a transcript derived from the 3'-terminal sequence of tobacco mosaic vir us RNA representing a minihelix capable of being enzymatically aminoac ylated with histidine. Imidazole buffer and spermine-imidazole conjuga tes in the presence of free imidazole cleave phosphodiester bonds in t he folded RNAs in a specific fashion. Imidazole buffer induces cleavag es preferentially in single-stranded regions because nucleotides in th ese regions have more conformational freedom and can assume more easil y the geometry needed for formation of the hydrolysis intermediate sta te. Spermine-imidazole constructs supplemented with free imidazole cle ave tRNA(Asp) within single-stranded regions after pyrimidine residues with a marked preference for pyrimidine-A sequences. Hydrolysis patte rns suggest a cleavage mechanism involving an attack by the imidazole residue of the electrostatically bound spermine-imidazole and by free imidazole at the most accessible single-stranded regions of the RNA. C leavages in a viral RNA fragment recapitulating a tRNA-like domain wer e found in agreement with the model of this molecule that accounts for its functional properties, thus illustrating the potential of the imi dazole-derived reagents as structural probes for solution mapping of R NAs. The cleavage reactions are simple to perform, provide information reflecting the state of the ribose-phosphate backbone of RNA and can be used for mapping single-and double-stranded regions in RNAs.