Determination of nucleotide distances in RNA by means of copper phenanthroline-generated hydroxyl radical cleavage pattern.
In contrast to the commonly used Fe(II)-EDTA, bis(orthophenanthroline)-copper(I) (OP-Cu) first generates hydroxyl radicals after binding to RNA. Due to diffusion, the hydroxyl radicals can cleave neighboring nucleotides in a distance r of up to 1.5 nm to the OP-Cu binding site. Using the known structure of tRNAPhe as a reference, we show that the hydroxyl radical cleavage pattern generated by a specifically bound OP-Cu shows a 1/r dependence on the distance of the cleaved nucleotide to the OP-Cu binding site. We propose that OP-Cu is a suitable probe for obtaining data on the distances between nucleotides in RNA, which can be used in modeling the structure of the examined RNA. However, this information is restricted to about three to four bases surrounding an OP-Cu binding site.
Hermann
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T
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Heumann
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H
D000926:Anticodon / Q000737:chemistry; D001665:Binding Sites; D017665:Hydroxyl Radical / Q000737:chemistry; D008958:Models, Molecular; D015335:Molecular Probes; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry; D010618:Phenanthrolines; D012313:RNA / Q000737:chemistry*; D012331:RNA, Fungal / Q000737:chemistry; D012360:RNA, Transfer, Phe / Q000737:chemistry; D012441:Saccharomyces cerevisiae; D013816:Thermodynamics
D000926:Anticodon; D015335:Molecular Probes; D009711:Nucleotides; D010618:Phenanthrolines; D012331:RNA, Fungal; D012360:RNA, Transfer, Phe; D017665:Hydroxyl Radical; D012313:RNA; C025205:1,10-phenanthroline