Oxidative crosslinking between tRNA and Protein
Authors:Bridgett Cabrera , Pia Cartagena, Evelyn Khamou , Kelsey Miller, Zitadel Anne Perez
Mentor:Eric Stemp, Department Chair, Physical Sciences, Mount St. Mary's College
Oxidation of guanine in DNA is known to lead to DNA-protein crosslinking. Transfer RNA molecules encounter many proteins during translation and we investigated whether guanine oxidation can also produce RNA-protein cross-linking. In a model system, tRNAPhe in the presence of histone or poly(lysine) was subjected to photochemical oxidation by an intercalator using the flash-quench technique, a method known to selectively oxidize guanine in double-stranded DNA. A number of different intercalators were tried but only two were successful. Irradiation of ethidium or Ru(phen)2dppz2+ [phen = phenanthroline, dppz = dipyridophenazine] in the presence of Co(NH3)5Cl2+ (an oxidative quencher) led to a loss in the intensity of the free band for the tRNA and a corresponding appearance of bands with lesser mobility in gel shift experiments. In addition, in the chloroform extraction assay, a drop in the 260 nm absorbance of the RNA was observed upon flash quench treatment. Both of these results are consistent with RNA-protein cross-linking as a result of guanine oxidation and suggest that this may be another consequence of oxidative stress.