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PMID: 2179949 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Purification and lipid-layer crystallization of yeast RNA polymerase II.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 87 ·No. 6 ·1990-03-00 ·Pages 2122-6

Edwards AM, Darst SA, Feaver WJ, Thompson NE, Burgess RR, Kornberg RD

Abstract

Yeast RNA polymerase II was purified to homogeneity by a rapid procedure involving immunoaffinity chromatography. The purified enzyme contained 10 subunits, as reported for conventional preparations, but with no detectable proteolysis of the largest subunit. In assays of initiation of transcription at the yeast CYC1 promoter, the enzyme complemented the deficiency of an extract from a strain that produces a temperature-sensitive polymerase II. Mammalian RNA polymerase II was inactive in this initiation assay. The purified yeast enzyme formed two-dimensional crystals on positively charged lipid layers, as previously found for Escherichia coli RNA polymerase holoenzyme. Image analysis of electron micrographs of crystals in negative stain, which diffracted to about 30-A resolution, showed protein densities of dimensions consistent with those of single polymerase molecules.

MeSH Terms
Chromatography, Affinity Crystallization Electrophoresis, Gel, Two-Dimensional Kinetics Lipid Bilayers Macromolecular Substances Microscopy, Electron Phosphatidylcholines RNA Polymerase II/isolation & purification,metabolism Saccharomyces cerevisiae/enzymology Transcription, Genetic
Chemicals
Lipid Bilayers Macromolecular Substances Phosphatidylcholines RNA Polymerase II
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Edwards A M
Department of Cell Biology, Stanford School of Medicine, CA 94305.
Darst S A
Feaver W J
Thompson N E
Burgess R R
Kornberg R D
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.87.6.2122
Published
1990-03-00
Pages
2122-6
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC53638
Subset
IM
Grants
NCI NIH HHS · 5-P01-CA23076-10 · United States
NIAID NIH HHS · AI21144 · United States
NIGMS NIH HHS · GM36659 · United States
External Links
PubMed source DOI: 10.1073/pnas.87.6.2122 PMC Full Text View journal details
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