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.
Gott
J M
JM
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Willis
M C
MC
Koch
T H
TH
Uhlenbeck
O C
OC
D001483:Base Sequence; D001665:Binding Sites; D001976:Bromouracil / Q000031:analogs & derivatives; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D012313:RNA / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D016601:RNA-Binding Proteins; D014466:Ultraviolet Rays; D014529:Uridine / Q000031:analogs & derivatives* / Q000378:metabolism
D036022:Capsid Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D016601:RNA-Binding Proteins; D001976:Bromouracil; D012313:RNA; C006824:5-bromouridine; D014529:Uridine