The Recruitment of the Saccharomyces cerevisiae Paf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex

Abstract

cTranscription elongation factors associate with RNA polymerase II and aid its translocation through chromatin. One such factor is the conserved Paf1 complex (Paf1C), which regulates gene expression through several mechanisms, including the stimulation of cotranscriptional histone modifications. Previous studies revealed a prominent role for the Rtf1 subunit in tethering Paf1C to the RNA polymerase II elongation machinery. Here, we investigated the mechanism by which Rtf1 couples Paf1C to active chromatin. We show that a highly conserved domain of Rtf1 is necessary and sufficient for mediating a physical interaction between Rtf1 and the essential transcription elongation factor Spt5. Mutations that alter this Rtf1 domain or delete the Spt5 C-terminal repeat domain (CTR) disrupt the interaction between Rtf1 and Spt5 and release Paf1C from chromatin. When expressed in cells as the only source of Rtf1, the Spt5-interacting domain of Rtf1 can associate independently with active genes in a pattern similar to that of full-length Rtf1 and in a manner dependent on the Spt5 CTR. In vitro experiments indicate that the interaction between the Rtf1 Spt5-interacting domain and the Spt5 CTR is direct. Collectively, our results provide molecular insight into a key attachment point between Paf1C and the RNA polymerase II elongation machinery.

Characterization of the Human Transcription Elongation Factor Rtf1: Evidence for Nonoverlapping Functions of Rtf1 and the Paf1 Complex

“…In S. cerevisiae, Rtf1 binds to Spt5 and serves as a binding platform for the PAF1C (37)(38)(39), and Cdc73 is required for the efficient recruitment of Rtf1 (2,47). Considering the weak physical interaction between Rtf1 and the PAF1C in metazoans, however, whether such reciprocal recruitment of Rtf1 and the PAF1C is conserved across species is questionable.…”

“…It can be said, however, that TFIIS is not responsible for the coactivator activity found in P0.3 D0.3. Considering the tight functional link between Rtf1 and chromatin (9,14,34,38), histones could modulate the transactivation potential of Rtf1 in vitro. Immunoblot analysis showed, however, that P0.3 D0.3 had an undetectable level of histones H2B and H3, and most of the histones were found to be depleted at the phosphocellulose column chromatography step (Fig.…”

“…In in vitro transcription assays using S. cerevisiae cell extracts and a naked DNA template, Rondon et al (16) showed that paf1⌬ and cdc73⌬ cell extracts were defective in elongation, whereas rtf1⌬ and leo1⌬ cell extracts were not; however, recent papers have shown that S. cerevisiae Rtf1 is critical for the recruitment of the PAF1C (20,34,35). The conserved Plus3 domain of Rtf1 interacts with the phosphorylated form of Spt5, the large subunit of DSIF, and thereby recruits the other PAF1C subunits to actively transcribed genes (5,(35)(36)(37)(38)(39). In higher eukaryotes, we used a human in vitro transcription system to show that the PAF1C cooperates with DSIF and Tat-SF1 to promote transcription elongation on a naked DNA template (13).…”

Structural basis for Spt5-mediated recruitment of the Paf1 complex to chromatin

“…Paf1C colocalizes with RNA pol II from the promoter to the 3′ end of genes (8,9) and mediates a diverse set of events, including transcription-coupled histone modifications (10)(11)(12)(13), phosphorylation of the RNA pol II C-terminal domain (CTD) (14,15), recruitment of termination and RNA 3′-end-processing factors (15)(16)(17), and maintenance of chromatin during transcription (18). Proper transcription-coupled ubiquitylation of histone H2B at lysine 123 in yeast is dependent on Paf1C recruitment to chromatin (19). Phenotypes caused by mutations that dissociate Paf1C from active genes argue that the ability of Paf1C to carry out its diverse set of activities in a controlled manner requires proper recruitment of Paf1C to actively transcribing RNA pol II (19)(20)(21)(22).…”

“…Proper transcription-coupled ubiquitylation of histone H2B at lysine 123 in yeast is dependent on Paf1C recruitment to chromatin (19). Phenotypes caused by mutations that dissociate Paf1C from active genes argue that the ability of Paf1C to carry out its diverse set of activities in a controlled manner requires proper recruitment of Paf1C to actively transcribing RNA pol II (19)(20)(21)(22).…”