An RNA-protein contact determined by 5-bromouridine substitution, photocrosslinking and sequencing.
An analogue of the replicase translational operator of bacteriophage R17, that contains a 5-bromouridine at position -5 (RNA 1), complexes with a dimer of the coat protein and photocrosslinks to the coat protein in high yield upon excitation at 308 nm with a xenon chloride excimer laser. Tryptic digestion of the crosslinked nucleoprotein complex followed by Edman degradation of the tryptic fragment bearing the RNA indicates crosslinking to tyrosine 85 of the coat protein. A control experiment with a Tyr 85 to Ser 85 variant coat protein showed binding but no photocrosslinking at saturating protein concentration. This is consistent with the observation from model compound studies of preferential photocrosslinking of BrU to the electron rich aromatic amino acids tryptophan, tyrosine, and histidine with 308 nm excitation.
Willis
M C
MC
Department of Chemistry, University of Colorado, Boulder 80309-0215.
LeCuyer
K A
KA
Meisenheimer
K M
KM
Uhlenbeck
O C
OC
Koch
T H
TH
D000595:Amino Acid Sequence; D001483:Base Sequence; D001976:Bromouracil / Q000031:analogs & derivatives; D002213:Capsid / Q000737:chemistry* / Q000378:metabolism; D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D017421:Sequence Analysis; D012694:Serine / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry*; D014466:Ultraviolet Rays; D014529:Uridine / Q000031:analogs & derivatives* / Q000737:chemistry
D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014443:Tyrosine; D012694:Serine; D001976:Bromouracil; D012324:RNA-Dependent RNA Polymerase; C006824:5-bromouridine; D014529:Uridine