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Nucleic Acids Research, Vol 24, Issue 17 3399-3406, Copyright © 1996 by Oxford University Press

ARTICLES

Fe.bleomycin as a probe of RNA conformation

CE Holmes, AT Abraham, SM Hecht, C Florentz and R Giege
Department of Chemistry, University of Virginia, Charlottesville 22901, USA.

Two crystallographically defined tRNAs, yeast tRNAAsp and tRNAPhe, were used as substrates for oxidative cleavage by Fe.bleomycin to facilitate definition at high resolution of the structural elements in RNAs conducive to bleomycin binding and cleavage. Yeast tRNAAsp underwent cleavage at G45 and U66; yeast tRNAPhe was cleaved at four sites, namely G19, A31, U52 and A66. Only two of these six sites involved oxidative cleavage of a 5'-G.Pyr-3' sequence, but three sites were at the junction between single- and double-stranded regions of the RNA, consistent with a binding model in which the bithiazole + C-terminal 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 elements in common with the mature tRNAs. Cleavage of these tRNAs by Fe.bleomycin gave patterns of cleavage very different from each other and than those of the mature tRNAs. This observation suggests strongly that Fe.bleomycin cannot be used for chemical mapping in the same fashion as more classical reagents, such as Pb2+ or dimethyl sulfate. However, the great sensitivity of Fe.bleomycin to changes in nucleic acid structure argues that those species which do show similar patterns of cleavage must be very close in structure.
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