Nucleic Acids Research, Vol 26, Issue 6 1421-1426, Copyright © 1998 by Oxford University Press
JJ Tate, J Persinger and B Bartholomew
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.
ARTICLES
Survey of four different photoreactive moieties for DNA photoaffinity labeling of yeast RNA polymerase III transcription complexes
Department of Medical Biochemistry, School of Medicine, Southern Illinois University at Carbondale, Carbondale, IL 62901-4413, USA.
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