Photoaffinity labeling of a promoter dependent eukaryotic transcription complex

Conference · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5019608

Meares, C F; Dahmus, M E

Photoaffinity labeling of Hela Cell RNA polymerase II transcription complexes has been done using thiouridine as a photoactivated crosslinker between RNA and protein. The nucleotide 4-thiouridine triphosphate is used as substrate by RNA polymerase II in the formation of promoter specific RNA transcripts. The length of transcript is controlled by addition of base specific RNA chain terminators, such as 3'-O-methyl guanosine triphosphate. The extract and DNA used are Hela cell S-100 purified through heparin Sepharose and a restriction fragment containing the major late promoter of adenovirus 2. After crosslinking, radiolabeled components are separated by polyacrylamide gel electrophoresis, blotted on to nitrocellulose, and visualized by autoradiography. Using this method of labeling the major proteins involved in transcription, they have been able to detect specific labeling of proteins corresponding in mobility to subunits IIo and IIc. Photoaffinity labeling is sensitive to low concentrations of alpha amanitin and requires both the addition of exogeneous DNA and 4-thiouridine.

• Research Organization: Univ. of California, Davis
• OSTI ID: 5019608
• Report Number(s): CONF-8606151-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
AROMATICS
AUTORADIOGRAPHY
AZAARENES
CHEMICAL REACTIONS
CROSS-LINKING
ENZYMES
GUANOSINE
HELA CELLS
HETEROCYCLIC COMPOUNDS
ISOTOPES
LABELLING
NUCLEIC ACIDS
NUCLEOSIDES
NUCLEOTIDES
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
POLYMERIZATION
PROTEINS
PURINES
RADIOISOTOPES
RIBOSIDES
RNA
RNA POLYMERASES
TRANSCRIPTION
TRANSFERASES