A specific, UV-induced RNA-protein cross-link using 5-bromouridine-substituted RNA.
The well-characterized RNA binding site of the bacteriophage R17 coat protein has been used to investigate the cross-linking of protein to 5-bromouridine (BrU)-substituted RNA using medium-wavelength UV light. We have demonstrated a specific RNA-protein cross-link and identified the site on the RNA of protein attachment. Formation of the covalent complex is dependent upon the presence of BrU at position -5 of the RNA and specific binding of the RNA by coat protein. The amount of cross-linking increases with time and depends on the light source and conditions used. Irradiations using a broad-spectrum UV transilluminator (peak at 312 nm) or monochromatic XeCl excimer laser (308 nm) gave levels of cross-linking exceeding 20 and 50%, respectively. The quantum yield of photo-cross-linking, determined with 308-nm excitation, was 0.003. While little strand breakage or debromination of the RNA occurred, significant protein photodamage was observed.[1]References
- A specific, UV-induced RNA-protein cross-link using 5-bromouridine-substituted RNA. Gott, J.M., Willis, M.C., Koch, T.H., Uhlenbeck, O.C. Biochemistry (1991) [Pubmed]
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