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Nucleic Acids Research, 1984, Vol. 12, No. 7 3405-3424
© 1984

MOLECULAR BIOLOGY

Mapping of psoralen cross-linked nucleotides in RNA

Esther Garrett-Wheeler, Raymond E. Lockard and Ajit Kumar

Department of Biochemistry, George Washington University, Medical School Washington, DC 20037, USA

Received November 22, 1983. Revised March 12, 1984. Accepted March 12, 1984.

A method is described for using the cross-linking reagent 4'-(hydroxy-methyl)-4,5',8-trimethylpsoralen (HMT) to map bsss 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 ribo-nuclease 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.


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