Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking.
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.
Tardiff
Daniel F
DF
Howard Hughes Medical Institute, Department of Biology, Brandeis University, 415 South Street, Waltham, MA 02454, USA.
Abruzzi
Katharine C
KC
Rosbash
Michael
M
D003432:Cross-Linking Reagents; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012319:RNA Polymerase II / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry; D015533:Transcriptional Activation / Q000235:genetics
D003432:Cross-Linking Reagents; D005656:Fungal Proteins; D021122:Protein Subunits; D016601:RNA-Binding Proteins; C082384:RNP1 protein, S cerevisiae; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; D012319:RNA Polymerase II; C511736:RPB1 protein, S cerevisiae; C112110:RPB3 protein, S cerevisiae