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

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.