Development of DNA affinity techniques for the functional characterization of purified RNA polymerase II transcription factors

Conference · Fri May 01 00:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6156265

Garfinkel, S; Thompson, J A; Cohen, R B; Brendler, T; Safer, B

Affinity adsorption, precipitation, and partitioning techniques have been developed to purify and characterize RNA Pol II transcription components from whole cell extracts (WCE) (HeLa) and nuclear extracts (K562). The titration of these extracts with multicopy constructs of the Ad2 MLP but not pUC8, inhibits transcriptional activity. DNA-binding factors precipitated by this technique are greatly enriched by centrifugation. Using this approach, factors binding to the upstream promoter sequence (UPS) of the Ad2 MLP have been rapidly isolated by Mono Q, Mono S, and DNA affinity chromatography. By U.V. crosslinking to nucleotides containing specific TSP-phosphodiester bonds within the recognition sequence, this factor is identified as a M/sub r/ = 45,000 polypeptide. To generate an assay system for the functional evaluation of single transcription components, a similar approach using synthetic oligonucleotide sequences spanning single promoter binding sites has been developed. The addition of a synthetic 63-mer containing the UPS element of the Ad2 MLP to HeLa WCE inhibited transcription by 60%. The addition of partially purified UPS binding protein, but not RNA Pol II, restored transcriptional activity. The addition of synthetic oligonucleotides containing other regulatory sequences not present in the Ad2 MLP was without effect.

• Research Organization: National Institutes of Health, Bethesda, MD
• OSTI ID: 6156265
• Report Number(s): CONF-870644-; TRN: 87-028709
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
RNA POLYMERASES
CHEMICAL PROPERTIES
FRACTIONATION
AFFINITY
CHROMATOGRAPHY
DNA
PHOSPHORUS 32
TRACER TECHNIQUES
TRANSCRIPTION
ULTRACENTRIFUGATION
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CENTRIFUGATION
DAYS LIVING RADIOISOTOPES
ENZYMES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RADIOISOTOPES
SEPARATION PROCESSES
TRANSFERASES
550201* - Biochemistry- Tracer Techniques