RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach.
To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3' end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.
Krogan
Nevan J
NJ
Banting and Best Department of Medical Research. Department of Molecular and Medical Genetics, University of Toronto. TYPO, Toronto Yeast Proteomics Organization, Toronto, Ontario, Canada.
Kim
Minkyu
M
Ahn
Seong Hoon
SH
Zhong
Guoqing
G
Kobor
Michael S
MS
Cagney
Gerard
G
Emili
Andrew
A
Shilatifard
Ali
A
Buratowski
Stephen
S
Greenblatt
Jack F
JF
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D056488:Histone Chaperones; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010445:Peptide Elongation Factors / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D020543:Proteome; D012319:RNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism; D035165:Transcription Factors, General; D019473:Transcription Factors, TFII; D035602:Transcriptional Elongation Factors
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D005656:Fungal Proteins; D056488:Histone Chaperones; D009687:Nuclear Proteins; C106768:POB3 protein, S cerevisiae; D010445:Peptide Elongation Factors; D010750:Phosphoproteins; D020543:Proteome; C071238:SPT16 protein, S cerevisiae; C080991:SPT4 protein, S cerevisiae; C065456:SPT6 protein, S cerevisiae; C489489:SUPT16H protein, human; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D035165:Transcription Factors, General; D019473:Transcription Factors, TFII; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; C069940:SPT5 transcriptional elongation factor; D012319:RNA Polymerase II; C056604:transcription factor TFIIF