Single base bulges in small RNA hairpins enhance ethidium binding and promote an allosteric transition.
A set of four RNA hairpin helices has been prepared by in vitro transcription with T7 RNA polymerase. The hairpins all contain the same nine base pair helix, but with an extra A, C, or U residue forming a bulge at one position; the fourth hairpin is a perfect helix with no bulge. The helix with a bulged A duplicates six base pairs of a helix in the 16S rRNA known to have an unusually high affinity for ethidium bromide [J. M. Kean, S. A. White, and D. E. Draper, Biochemistry 24, 5062 (1985)]. Binding and chemical cleavage studies with ethidium or the reagent methidiumpropylEDTA-Fe(II) [MPE-Fe(II)] showed that the sequence CpG is a preferred intercalation site whether or not a bulge is present; all three bulged bases enhance intercalation at the CpG sequence by an order of magnitude; and intercalation in a bulged helix results in a concerted conformational change involving the entire helix backbone, again dependent on the presence of a bulge but independent of the particular base. These results suggest that an extra sugar-phosphate residue in an RNA helix backbone has a dramatic effect on the ability of the RNA to adopt new conformations. This effect could be an important reason for the conservation of bulges at certain positions in ribosomal and other RNAs.
D000495:Allosteric Site; D001667:Binding, Competitive; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D004996:Ethidium / Q000378:metabolism; D007364:Intercalating Agents / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000378:metabolism*
D007364:Intercalating Agents; C038200:methidiumpropyl-EDTA-iron(II); D012313:RNA; D004492:Edetic Acid; D004996:Ethidium