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

Abstract

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.The synthesis of functional mRNA precursors by eukaryotic RNA polymerase II (RNAPII) is a multistage process consisting of three major steps: initiation, elongation, and termination. Although much has been learned about steps involved in initiation, including formation of the initiation complex, initiation of the RNA chain, and promoter clearance, much less is known about later stages in transcription, especially chain elongation. It is known, however, that the transition from transcriptional initiation to elongation is associated with a change in the factors that are associated with RNAPII.Whereas general transcription factors are required for promoter-directed transcription initiation, and the multisubunit Srb/Mediator complex is associated with the RNAPII holoenzyme that binds to promoters in the yeast Saccharomyces cerevisiae, RNAPII can elongate the transcript unaided. However, several factors have been discovered that stimulate elongation on nonchromatin, "naked" DNA templates (e.g., TFIIS and TFIIF) (32,47). Recently, other factors have been identified that are thought to affect RNAPII transcript elongation via effects on chromatin. These include the yeast factors Spt4 and Spt5 (corresponding to human 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole [DRB] sensitivity-inducing factor [DSIF], which is req...

Architecture of the yeast Elongator complex

“…We constructed yeast strains carrying endogenously TAP‐tagged versions of Elp1 and Elp6 and purified Elongator using previously established purification protocols 20. Consistent with sub‐stoichiometric cellular amounts of Elp456 in vivo 28, 36, 37, purifications of Elp1‐TAP resulted in an excess of Elp123 sub‐complex 20, whereas Elp6‐TAP purifications resulted in reduced quantities but yielded highly pure, complete, and stoichiometric Elongator complex. Large‐scale preparations yielded sufficient amounts of pure Elongator complex and Elp123 sub‐complex to analyze their overall architecture and shape by EM.…”

Bromodomain Factor 1 (Bdf1) Is Phosphorylated by Protein Kinase CK2

“…Preparation of yeast whole-cell extracts and purification of TAP-tagged Bdf1 were performed essentially as described previously (17,27) with minor modifications. The BDF1-associated proteins were electrophoresed at 10 mA in a sodium dodecyl sulfate (SDS)-10% polyacrylamide gel and silver stained.…”

“…Basal transcription factors for both RNA Pol I (29) and Pol III (8,10,11) are substrates for CK2. Furthermore, CK2 copurifies with several chromatin-related complexes, including FACT (Spt16/ Pob3) and Chd1 (17). CK2 also regulates the activity of Fcp1, the RNA Pol II CTD phosphatase (6,25).…”

Structure and in Vivo Requirement of the Yeast Spt6 SH2 Domain

“…34 We expected similarity between these data sets, since Spt6 interacts genetically with TFIIS, 5 and with the Rtf1-containing Paf1 complex, 35,36 and since Spt6 binds the Spt4-Spt5 complex. 37 An unsupervised hierarchical cluster analysis showed that the differential expression data from the spt4Δ and rtf1Δ strains form a distinct cluster within a dendrogram, indicating similarity of their gene expression profiles (Fig. 6b, lanes 4 and 5, Materials and Methods).…”