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

Abstract

Polymerase associated factor 1 complex (Paf1C) broadly influences gene expression by regulating chromatin structure and the recruitment of RNA-processing factors during transcription elongation. The Plus3 domain of the Rtf1 subunit mediates Paf1C recruitment to genes by binding a repeating domain within the elongation factor Spt5 (suppressor of Ty). Here we provide a molecular description of this interaction by reporting the structure of human Rtf1 Plus3 in complex with a phosphorylated Spt5 repeat. We find that Spt5 binding is mediated by an extended surface containing phosphothreonine recognition and hydrophobic interfaces that interact with residues outside the Spt5 motif. Changes within these interfaces diminish binding of Spt5 in vitro and chromatin localization of Rtf1 in vivo. The structure reveals the basis for recognition of the repeat motif of Spt5, a key player in the recruitment of gene regulatory factors to RNA polymerase II.histone modification | scaffold | DSIF | Cdk9 | crystallography

Fission yeast RNA triphosphatase reads an Spt5 CTD code

“…The present structural snapshot of the RNA capping enzyme Pct1 bound to Spt5 CTD sequence TPAWN extends insights from the recently reported crystal structure of a human Spt5 CTR binding protein (Wier et al 2013): the Plus3 domain of Rtf1 bound to a phosphorylated CTR peptide SGSRT P PMYGSQ. Figure 6 shows an alignment of the Spt5 ligands from the two structures, superimposed at their respective Thr-Pro dipeptides.…”

“…The proline is in the trans conformation in both structures. In the case of Rtf1, the Spt5 interface is dominated by hydrogen bonding contacts to the phosphate group of the Spt5 ligand, consistent with the fact that threonine phosphorylation is required for Rtf1 binding to Spt5-CTR (Wier et al 2013). In contrast, Pct1 makes no contacts with the Spt5 threonine side chain and its binding to Spt5 is antagonized by threonine phosphorylation.…”

“…Available evidence suggests that threonine phosphorylation of Spt5 CTD repeats inscribes a binary code (on-off) that is read by diverse CTD receptors, each in its own distinctive manner. Whereas the Paf1 complex subunit Rtf1 recognizes only the phosphorylated Spt5 CTR (Wier et al 2013), the RNA guanylyltransferase Pce1 ) and the RNA triphosphatase Pct1 (present study) specifically bind to the unmodified form of the CTD. Moreover, the positive elongation function of Spt5 depends on phosphorylation of the CTD/CTR by Cdk9 (Yamada et al 2006).…”

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

“…2, the most likely candidate for the Rtf1 coactivator was the PAF1C. Based on a recent study in S. cerevisiae (35), DSIF might also be a candidate for the Rtf1 coactivator, although DSIF did not comigrate with the coactivator activity at the phosphocellulose step. To explore these possibilities, the PAF1C was immunodepleted from HeLa cell NE using antibodies against the PAF1C.…”

“…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).…”