Detection of high-affinity intercalator sites in a ribosomal RNA fragment by the affinity cleavage intercalator methidiumpropyl-EDTA-iron(II).
The affinity cleavage reagent methidiumpropyl-EDTA (MPE) [Hertzberg, R. P., & Dervan, P. B. (1982) J. Am. Chem. Soc. 104, 313-315] intercalates between base pairs in helical DNA and, when complexed with Fe(II), cleaves the DNA by oxidative degradation of the deoxyribose. We find that this reagent is useful for mapping structure in some RNA molecules. The reagent binds to poly(A)-poly(U) with the same or slightly lower affinity as the related ethidium intercalator, selectively binds double-helical in preference to single-stranded RNA, and when complexed with Fe(II) readily cleaves the RNA backbone. The reagent binds to three or four helical locations in tRNAPhe with an affinity of 10(5)-10(6) M-1 (0.1 M Na+, pH 7.6, 37 degrees C). With a 345-base RNA fragment covering the S8/S15 protein binding region of Escherichia coli 16S ribosomal RNA, MPE-Fe(II) intercalates strongly at two helical sites: one is located at or near a single base bulge and the other at the end of a helix. Intense cutting is also seen in a region that is not part of a Watson-Crick helix. Ethidium bromide binds at these sites with high affinity (about 10(7) M-1 at 0.1 M Na+, pH 7.6, 37 degrees C). The sites are all clustered in a region of the RNA thought to bind S15. Tertiary folding of the RNA may distort helices in the molecule to create sites with particularly high affinities for intercalators; such sites may have functional significance in protein recognition or RNA-RNA interactions.
D001483:Base Sequence; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D004996:Ethidium / Q000494:pharmacology; D007502:Iron Chelating Agents / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D012335:RNA, Ribosomal / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism
D007502:Iron Chelating Agents; D012335:RNA, Ribosomal; D012346:RNA, Transfer, Amino Acyl; C038200:methidiumpropyl-EDTA-iron(II); D004492:Edetic Acid; D004996:Ethidium