Genome-wide location analysis reveals a role of TFIIS in RNA polymerase III transcription

Abstract

TFIIS is a transcription elongation factor that stimulates transcript cleavage activity of arrested RNA polymerase II (Pol II). Recent studies revealed that TFIIS has also a role in Pol II transcription initiation. To improve our understanding of TFIIS function in vivo, we performed genome-wide location analysis of this factor. Under normal growth conditions, TFIIS was detected on Pol II-transcribed genes, and TFIIS occupancy was well correlated with that of Pol II, indicating that TFIIS recruitment is not restricted to NTP-depleted cells. Unexpectedly, TFIIS was also detected on almost all Pol III-transcribed genes. TFIIS and Pol III occupancies correlated well genome-wide on this novel class of targets. In vivo, some dst1 mutants were partly defective in tRNA synthesis and showed a reduced Pol III occupancy at the restrictive temperature. In vitro transcription assays suggested that TFIIS may affect Pol III start site selection. These data provide strong in vivo and in vitro evidence in favor of a role of TFIIS as a general Pol III transcription factor. Gene transcription is a complex and highly regulated process that, in eukaryotes, is carried out by three specialized RNA polymerases (Pol I, II, and III), dedicated to the transcription of different sets of genes. Transcription starts by the assembly of large preinitiation complexes (PIC) comprising TBP (the TATA-binding protein common to all three transcription systems) and general transcription factors specific to the RNA polymerase considered. Other transcription factors facilitate RNA elongation by Pol II (Shilatifard et al. 2003). One of these elongation factors, TFIIS, was initially discovered in human (Natori et al. 1973) and yeast cells (Sawadogo et al. 1980b), and is highly conserved in the eukaryotic and archaeal kingdoms (Hausner et al. 2000;Fish and Kane 2002). Structurally unrelated but functionally equivalent factors (GreA and GreB) also operate in bacteria (Borukhov et al. 1993;Opalka et al. 2003). TFIIS forms a binary complex with Pol II and stimulates an intrinsic transcript cleavage activity of RNA polymerase allowing elongating enzymes to resume RNA synthesis after accidental transcription arrest (Fish and Kane 2002;Kettenberger et al. 2003).TFIIS is organized in three domains as determined by limited proteolysis and nuclear magnetic resonance (NMR) (Awrey et al. 1997;Olmsted et al. 1998). The three-dimensional structure of the Pol II-TFIIS complex, without the TFIIS N-terminal domain, has been determined by crystallographic studies (Kettenberger et al. 2003). The Nterminal domain I of TFIIS protrudes from Pol II and is not required for elongation (Nakanishi et al. 1995;Awrey et al. 1998), but binds the yeast Mediator and SAGA coactivator (Wery et al. 2004). The TFIIS central domain II is inserted into the funnel-shaped pore of Pol II and brings the C-terminal conserved RSADE motif very close to the enzyme active site (Kettenberger et al. 2003). Remarkably, the C-terminal domain III of TFIIS shares the RSADE motif with the C-terminal parts o...

Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment

“…The spatial Rad2 distribution on the longest class III gene, SCR1, was similar to that of Pol III (Supplemental Fig. S2D; Ghavi-Helm et al 2008). We also confirmed the presence of Rad2 on telomeric regions (Supplemental Fig.…”

Genome-wide location analysis reveals a role for Sub1 in RNA polymerase III transcription

“…A conventional ChIP experiment on the rDNA locus (Fig. S2) confirmed that Sub1 (but not TFIIIC) bound all along the rDNA gene locus but preferentially to the Pol I-transcribed region, an enrichment profile similar to that of TFIIS, a protein involved in Pol II transcription that has been shown recently to play a role in Pol III transcription (19).…”

“…1C, Sub1 was detected significantly all along the SCR1 gene and over the tRNA Ileu gene, but not on the intergenic regions that separate these loci. A comparison of the enrichment profile of Sub1 across the SCR1 gene with that of TFIIIC, TFIIIB and Pol III (19) suggested a colocalization of Sub1 with Pol III.…”

The poly(A)-binding protein Nab2 functions in RNA polymerase III transcription

“…RNAPIII, TFIIIB, and TFIIIIC show the expected profiles (Supplemental Fig. S5; Ghavi-Helm et al 2008). Nab2 has its highest occupancies at the 5 ′ end of SCR1 (Supplemental Fig.…”