Probing the higher order structure of RNA with peroxonitrous acid

FEBS Letters

Ehresmann, Bernard¹  Gross, Hans J.³  Keith, Gérard¹  Isel, Catherine¹  Heumann, Hermann⁴  Götte, Matthias⁴  Marquet, Roland¹  Anderson, Vernon E.²  Ehresmann, Chantal¹ 

[1]UPR no 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15, rue R. Descartes, F-67084 Strasbourg Cedex, France

[2]Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA

[3]Bayerische Julius-Maximilians-Universität, Institut für Biochemie, Biozentrum, Am Hubland, D-97074 Würzburg, Germany

[4]Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany

关键词: Chemical probing;    Hydroxyl radicals;    Fe-EDTA;    ONOOK;    tRNA;   

DOI  :  10.1016/0014-5793(96)00662-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Potassium peroxinitrite (ONOOK) and [Fe(EDTA)]²⁻ were used to analyze the influence of chemically entirely different hydroxyl radical sources on tRNA cleavage profiles. [Fe(EDTA)]²⁻ gives rise to hydroxyl radicals via a Fenton-like reaction during the oxidation of chelated Fe²⁺, while ONOOK generates hydroxyl radicals via its conjugate acid (ONOOK) when adding a stable alkaline solution of ONOOK in samples buffered at neutral pH. [Fe(EDTA)]²⁻ is known to induce oxidative strand scission at sugar moieties thought to be solvent accessible, while those residues located in the ‘inside’ of structured RNAs are protected. Although ONOOH is neutral and significantly smaller than the metal complex, both reagents generate the same protection pattern on tRNAs, suggesting that access of the commonly formed hydroxyl radical, rather than access of its source, is the determining factor when probing the higher order structure of RNA. Strong difference in reactivity is only seen at the modified 2-thiouridine S34 of tRNA^Lys3 which shows hyperreactivity towards ONOOK treatment. This particular reaction may require interaction between the peroxonitrite anion and the thiocarbonyl group of the base, since hyperreactivity is not observed when probing the dethiolated tRNA^Lys3.

【 授权许可】

Unknown