Protocol

DNA-Protein Interactions

Volume 148 of the series Methods in Molecular Biology pp 363-381

Site-Directed DNA Photoaffinity Labeling of RNA Polymerase III Transcription Complexes

  • Jim PersingerAffiliated withDepartment of Biochemistry and Molecular Biology, School of Medicine, Southern Illinois University
  • , Blaine BartholomewAffiliated withDepartment of Biochemistry and Molecular Biology, School of Medicine, Southern Illinois University

* Final gross prices may vary according to local VAT.

Get Access

Abstract

Site-specific DNA photoaffinity labeling is a useful technique for mapping interactions of proteins with DNA in complex systems such as the yeast RNA polymerase III (Pol III) transcription complex, which consists of at least 25 different proteins (1,2). This technique allows probing of protein-DNA interactions across large stretches of DNA and can be done in relatively crude extracts. The regions or domains of the protein contacting DNA can be identified by peptide mapping of the photoaffinity labeled protein. Our discussion of DNA photoaffinity labeling will focus on (1) the synthesis of photoreactive nucleotide analogs, (2) the manner in which the photoreactive nucleotide is incorporated into DNA, and (3) experimental details of DNA photoaffinity labeling.