Pol II CTD kinases Bur1 and Kin28 promote Spt5 CTR-independent recruitment of Paf1 complex.
Paf1 complex (Paf1C) is a transcription elongation factor whose recruitment is stimulated by Spt5 and the CDKs Kin28 and Bur1, which phosphorylate the Pol II C-terminal domain (CTD) on Serines 2, 5, and 7. Bur1 promotes Paf1C recruitment by phosphorylating C-terminal repeats (CTRs) in Spt5, and we show that Kin28 enhances Spt5 phosphorylation by promoting Bur1 recruitment. It was unclear, however, whether CTD phosphorylation by Kin28 or Bur1 also stimulates Paf1C recruitment. We find that Paf1C and its Cdc73 subunit bind diphosphorylated CTD repeats (pCTD) and phosphorylated Spt5 CTRs (pCTRs) in vitro, and that cdc73 mutations eliminating both activities reduce Paf1C recruitment in vivo. Phosphomimetic (acidic) substitutions in the Spt5 CTR sustain high-level Paf1C recruitment in otherwise wild-type cells, but not following inactivation of Bur1 or Kin28. Furthermore, inactivating the pCTD/pCTR-interaction domain (PCID) in Cdc73 decreases Paf1C-dependent histone methylation in cells containing non-phosphorylatable Spt5 CTRs. These results identify an Spt5 pCTR-independent pathway of Paf1C recruitment requiring Kin28, Bur1, and the Cdc73 PCID. We propose that pCTD repeats and Spt5 pCTRs provide separate interaction surfaces that cooperate to ensure high-level Paf1C recruitment.
Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA.
Hongfang
H
Qiu
Cuihua
C
Hu
Naseem A
NA
Gaur
Alan G
AG
Hinnebusch
D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004257:DNA Polymerase II / Q000378:metabolism; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D035602:Transcriptional Elongation Factors / Q000378:metabolism*
D002868:Chromosomal Proteins, Non-Histone; D009687:Nuclear Proteins; C097698:PAF1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035602:Transcriptional Elongation Factors; C069940:SPT5 transcriptional elongation factor; D018844:Cyclin-Dependent Kinases; C414163:SGV1 protein, S cerevisiae; C084210:Kin28 protein kinase, S cerevisiae; D004257:DNA Polymerase II