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Proc Natl Acad Sci U S A. 2007 Dec 11;104(50):19948-53. Epub 2007 Dec 5.

Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking.

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  • 1Howard Hughes Medical Institute, Department of Biology, Brandeis University, 415 South Street, Waltham, MA 02454, USA.

Abstract

To characterize proteins associated with active transcription complexes, we purified RNA polymerase II (pol II) from Saccharomyces cerevisiae after fixing live cells with formaldehyde. The approach mimics ChIP and requires solubilizing cross-linked complexes with sonication. Pol II was affinity-purified, and associated proteins were identified by MS. Several classes of proteins depended on cross-linking, including Mediator, general transcription factors, elongation factors, ribonucleoprotein particle (RNP) proteins, and histones. A tagged RNP protein reciprocally purified pol II under identical cross-linking conditions, and the association between RNP proteins and pol II was largely RNase-sensitive. The data indicate that the cross-linked Pol II purification contains elongating pol II with associated nascent RNP. Consistent with this view, some elongation factors no longer associate with pol II after inactivation of transcription in the temperature-sensitive pol II mutant, rpb1-1. Taken together, our data suggest that the cross-linked pol II purification contains a mixed population of pol II, including initiating pol II and elongating pol II.

PMID:
18077427
[PubMed - indexed for MEDLINE]
PMCID:
PMC2148403
Free PMC Article

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