An RNA - protein contact determined by 5-bromouridine substitution, photocrosslinking and sequencing

Michael C. Willis, Karen A. Lecuyer, Kristen M. Meisenheimer, Olke C. Uhlenbeck, Tad H. Koch*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

43 Scopus citations

Abstract

An analogue of the replicase translational operator of bacteriophage R17, that contains a 5-bromouridine at position -5 (RNA I), 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.

Original languageEnglish (US)
Pages (from-to)4947-4952
Number of pages6
JournalNucleic acids research
Volume22
Issue number23
DOIs
StatePublished - Nov 25 1994

ASJC Scopus subject areas

  • Genetics

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