AN RNA-PROTEIN CONTACT DETERMINED BY 5-BROMOURIDINE SUBSTITUTION, PHOTO-CROSS-LINKING AND SEQUENCING

作者:WILLIS MC; LECUYER KA; MEISENHEIMER KM; UHLENBECK OC; KOCH TH
来源:Nucleic Acids Research, 1994, 22(23): 4947-4952.
DOI:10.1093/nar/22.23.4947

摘要

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.

  • 出版日期1994-11-25

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