TY - JOUR
T1 - Survey of four different photoreactive moieties for DNA photoaffinity labeling of yeast RNA polymerase III transcription complexes
AU - Tate, Jennifer J.
AU - Persinger, Jim
AU - Bartholomew, Blaine
N1 - Funding Information:
We would like to thank Julie Ellis for her help in the synthesis and characterization of the photoreactive deoxyuridine analogs and to Alexander Kogon for DB-NHS and technical advice. This work was supported by Public Health Service grant GM 48413 from the NIHGMS.
PY - 1998/3/15
Y1 - 1998/3/15
N2 - 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 tRNA(Tyr) 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.
AB - 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 tRNA(Tyr) 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.
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U2 - 10.1093/nar/26.6.1421
DO - 10.1093/nar/26.6.1421
M3 - Article
C2 - 9490787
AN - SCOPUS:0032520692
SN - 0305-1048
VL - 26
SP - 1421
EP - 1426
JO - Nucleic acids research
JF - Nucleic acids research
IS - 6
ER -