Survey of four different photoreactive moieties for DNA photoaffinity labeling of yeast RNA polymerase III...

In order to optimize the detection of protein-DNA contacts by DNA photoaffinity labeling, we attached four different photoreactive groups to DNA and examined their ability to crosslink yeast RNA polymerase III (Pol III) transcription complexes. Photoreactive nucleotides containing an aryl azide (AB-dUMP), benzophenone (BP-dUMP), perfluorinated aryl azide (FAB-dUMP) or diazirine (DB-dUMP) coupled to 5-aminoallyl deoxyuridine were incorporated into the SUP4 tRNATyr gene at bp -3/-2 or +11. Photo-crosslinking with diazirine revealed contacts of Pol III with DNA that are not detected by DNA photoaffinity labeling using an aryl azide, fluorinated aryl azide or benzophenone group attached to DNA. These novel contacts were of the 82 kDa subunit of Pol III with DNA at bp -3/-2 in the initiation complex and of the 82, 40(37) and 31 kDa subunits of Pol III with DNA at bp +11 in elongation complexes stalled at bp +17. These results provide evidence for the subcomplex of the 82, 34 and 31 kDa subunits of Pol III being positioned near the transcription bubble of actively transcribing Pol III, as all three proteins were crosslinked at bp +11 of the stalled transcription complex.