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Molecular and Cellular Biology, October 2002, p. 6979-6992, Vol. 22, No. 20
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.20.6979-6992.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

RNA Polymerase II Elongation Factors of Saccharomyces cerevisiae: a Targeted Proteomics Approach

Nevan J. Krogan,1,2,3 Minkyu Kim,4 Seong Hoon Ahn,4 Guoqing Zhong,1 Michael S. Kobor,1,2 Gerard Cagney,1,3 Andrew Emili,1,2,3 Ali Shilatifard,5 Stephen Buratowski,4 and Jack F. Greenblatt1,2,3*

Banting and Best Department of Medical Research,1 Department of Molecular and Medical Genetics, University of Toronto,2 TYPO, Toronto Yeast Proteomics Organization, Toronto, Ontario, Canada,3 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115,4 Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 631045

Received 20 May 2002/ Returned for modification 20 June 2002/ Accepted 16 July 2002

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.

* Corresponding author. Mailing address: Banting and Best Department of Medical Research, University of Toronto, 112 College St., Rm. 210, Toronto, Ontario, Canada M5G 1L6. Phone: (416) 978-4141. Fax: (416) 978-8528. E-mail: jack.greenblatt{at}utoronto.ca.

Molecular and Cellular Biology, October 2002, p. 6979-6992, Vol. 22, No. 20
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.20.6979-6992.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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