FE BLEOMYCIN AS A PROBE OF RNA CONFORMATION

Two crystallographically defined tRNAs, yeast tRNA(Asp) and tRNA(Phe), were used as substrates for oxidative cleavage by Fe bleomycin to fac ilitate definition at high resolution of the structural elements in RN As conducive to bleomycin binding and cleavage. Yeast tRNA(Asp) underw ent cleavage at G(45) and U-66; yeast tRNA(Phe) was cleaved at four si tes, namely G(19), A(31), U-52 and A(66) Only two of these six sites i nvolved oxidative cleavage of a 5'-G Pyr-3' sequence, but three sites were at the junction between single- and double-stranded regions of th e RNA, consistent with a binding model in which the bithiazole + C-ter minal substituent of bleomycin bind to minor groove structures on the RNA, Also studied were four tRNA transcripts believed on the basis of biochemical and chemical mapping experiments to share structural eleme nts in common with the mature tRNAs. Cleavage of these tRNAs by Fe ble omycin gave patterns of cleavage very different from each other and th an those of the mature tRNAs, This observation suggests strongly that Fe bleomycin cannot be used for chemical mapping in the same fashion a s more classical reagents, such as Pb2+ or dimethyl sulfate, However, the great sensitivity of Fe bleomycin to changes in nucleic acid struc ture argues that those species which do show similar patterns of cleav age must be very close in structure.