Mapping of psoralen cross-linked nucleotides in RNA.
A method is described for using the cross-linking reagent 4'-(hydroxy-methyl)-4,5',8-trimethylpsoralen (HMT) to map base paired regions and higher-order structure within RNA molecules. Applying this method to yeast tRNAPhe, we have specifically identified cross-links within the acceptor stem between U6 X U68, in the D-stem between C11 X C25, and in the T psi-stem between U50 X C63 and U52 X C63. We have also identified a unique cross-link between U8 X C48 which are trans pyrimidines in the core region due to tertiary interactions between U8:A14 and C48:G15. The precise point of cross-linking was deduced in every case by using purine-specific U2 ribonuclease along with cytidine-specific CL3 ribonuclease which will anomalously cleave after photoreversed pyrimidines. The ability to map the precise point of cross-linking should prove invaluable in identifying nucleotides in close proximity within the tertiary structure of other RNA molecules.
D001482:Base Composition; D001483:Base Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D003432:Cross-Linking Reagents; D011564:Furocoumarins; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides; D010761:Phosphorus Radioisotopes; D012346:RNA, Transfer, Amino Acyl; D006163:Ribonuclease T1; D014307:Trioxsalen / Q000031:analogs & derivatives
D003432:Cross-Linking Reagents; D011564:Furocoumarins; D009843:Oligoribonucleotides; D010761:Phosphorus Radioisotopes; D012346:RNA, Transfer, Amino Acyl; C016507:hydroxymethyltrioxsalen; D006163:Ribonuclease T1; D014307:Trioxsalen