Analyses of Protein-Nucleic Acid complexes by MS - Reactivity of Various Amino Acids with Nucleotides upon UV Irradiation

摘要

A comprehensive mass spectrometric analysis on cross-linked native ribonucleoprotein complexes and in vitro reconstituted RNPs identified amino acids: cysteine, histidine, leucine, lysine, methionine, phenylalanine, and tyrosine cross-linked to nonsubstituted uracil; lysine and proline are reactive towards 4thioU and phenylalanine and leucine reactive towards 6thioG. Loss of H_2S was observed in the UV induced reaction of substituted bases, in contrast, non-substituted RNA shows mainly an additive behavior of the components (except of cysteine where the cross-linking reaction is accompanied by addition of an extra mass of 152 amu). We believe that the different cross-links of the amino acids to the various substituted RNA bases reflect also their stability when probing cross-linked RNA with reverse transcriptase reaction (that translated RNA into DNA) like in CLIP and PAR-CLIP molecular biology approaches.