Oxidative Crosslinking between tRNA and Protein
Authors:Pia Cartagena, Bridgett Gabrera, Kelsey Miller, Alyson Thien, Alyson Thien
Mentor:Eric Stemp, Professor of Chemistry , Mount St. Mary's College
Oxidation of guanine can lead to DNA-protein crosslinking. Transfer RNA molecules encounter many proteins during translation and we investigated whether guanine oxidation can also produce RNA-protein crosslinking. Oxidative damage to RNA can result in aging, cancer, and many other diseases. These crosslinks and formation of guanine radicals tRNAPhe in the presence of histone or poly(lysine) was subjected to the flash-quench technique, a method known to selectively oxidize guanine. 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. 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. The emission intensity of the Y-base from tRNA was also found to decrease over time which suggests that the Y-base can be oxidized using the flash quench technique.