Random-splitting of tRNA transcripts as an approach for studying tRNA-protein interactions

Location of phosphodiester bonds essential for aminoacylation of bovine tRNA(Trp) was identified using a randomly cleaved transcript synthesized in vitro. It was found that cleavage of phosphodiester bonds after nucleotides in positions 21, 22, 36-38, 57-59, 62 and 64 were critical for aminoacylation capacity of tRNA(Trp)-transcript. These cleavage sites were located in the regions of tRNA molecule protected by the cognate synthetase against chemical modification and in the regions presumably outside the contact area as well. These results indicate that for maintenance of aminoacylation ability the intactness of the certain regions of the tRNA backbone structure is necessary. Random splitting of non-modified RNA with alkali followed by separation of active and inactive molecules and identification of cleavage sites developed in this work may become a general approach for studying the role of RNA covalent structure in its interaction with proteins.