Ctr9, Rtf1, and Leo1 are components of the Paf1/RNA polymerase II complex

Abstract

The Saccharomyces cerevisiae Paf1-RNA polymerase II (Pol II) complex is biochemically and functionally distinct from the Srb-mediator form of Pol II holoenzyme and is required for full expression of a subset of genes. In this work we have used tandem affinity purification tags to isolate the Paf1 complex and mass spectrometry to identify additional components. We have established that Ctr9, Rtf1, and Leo1 are factors that associate with Paf1, Cdc73, and Pol II, but not with the Srb-mediator. Deletion of either PAF1 or CTR9 leads to similar severe pleiotropic phenotypes, which are unaltered when the two mutations are combined. In contrast, we found that deletion of LEO1 or RTF1 leads to few obvious phenotypes, although mutation of RTF1 suppresses mutations in TATA-binding protein, alters transcriptional start sites, and affects elongation. Remarkably, deletion of LEO1 or RTF1 suppresses many paf1Delta phenotypes. In particular, an rtf1Delta paf1Delta double mutant grew faster, was less temperature sensitive, and was more resistant to caffeine and hydroxyurea than a paf1Delta single mutant. In addition, expression of the G(1) cyclin CLN1, reduced nearly threefold in paf1Delta, is restored to wild-type levels in the rtf1Delta paf1Delta double mutant. We suggest that lack of Paf1 results in a defective complex and a block in transcription, which is relieved by removal of Leo1 or Rtf1.

FIG. 1.

The Paf1 complex is distinct from the Srb-Med complex. Whole-cell extracts made from a TAP-tagged Cdc73 strain (YJJ1307) and a TAP-tagged Srb5 strain (YJJ1308) were incubated with rabbit IgG agarose. Unbound proteins were washed away, and proteins that associate with TAP-Cdc73 were eluted by the addition of TEV protease. Fractions (IP, input; FT, flowthrough; E, eluate) were loaded onto a 4-to-12% NuPAGE gel and analyzed by Western blotting with antibodies as indicated. The asterisk indicates cross-reacting IgG from the IgG agarose column, and the arrow indicates the TEV protease cleaved form of TAP-Srb5. We observe two cross-reacting bands with the α-Paf1 antibody (see also Fig. 5B and reference 47); however, the faint band indicated by the open circle in the TAP-tagged Srb5 eluate does not correspond to either form of Paf1, and its presence is not reproducible (see Fig. 5B).

FIG. 2.

The Paf1 complex is approximately 1.7 MDa. Proteins that associate with Cdc73-TAP after chromatography on IgG agarose and elution with TEV protease were subjected to gel filtration chromatography on a Superose 6 gel filtration column. Fractions were separated on a 4-to-12% NuPAGE gel and analyzed by Western blotting with the indicated antibodies. Sizes were determined by calibrating the gel filtration column with the indicated standards as described in Materials and Methods.

FIG. 3.

Ctr9, Rtf1, Leo1, and Spt5 associate with the Paf1 complex. Fractions 37 to 41 and 69 to 73 from the Superose 6 gel filtration column were pooled, TCA precipitated, and separated on a 4-to-12% NuPAGE gel. Protein bands were visualized by colloidal blue staining and processed for MALDI-TOF analysis. Proteins that were accurately identified are marked.

FIG. 4.

Both Ctr9 and Hpr1 associate with RNA Pol II and Paf1. (A) Ctr9-associated proteins were purified from a TAP-tagged Ctr9 strain (YJJ1329). Samples were fractionated on a Superose 6 gel filtration column and immunoblotted with the indicated antibodies. (B) Hpr1-associated proteins were purified from a TAP-tagged Hpr1 strain (YJJ1334). Samples were fractionated on a gel filtration column and immunoblotted with the indicated antibodies.

FIG. 5.

Leo1-HA interacts with the Paf1 complex but not with the Srb-Med complex. (A) Coimmunoprecipitation of Leo1-HA with Paf1 and Rtf1. Whole-cell extracts prepared from the wild type (YJJ662) or a Leo1-HA strain (YJJ1330) were subjected to immunoprecipitation with an anti-HA (12CA5) antibody as described in Materials and Methods. Immunoprecipitates were analyzed by Western blotting using the indicated antibodies. IP, input; UB, unbound fraction; B, bound fraction. (B) TAP tag purification of Leo1-HA. Whole-cell extracts prepared from strains containing both a TAP tag and a Leo1-HA tag (YJJ1309, YJJ1331, and YJJ1332) were incubated with IgG beads as described in Materials and Methods. After washing, proteins were eluted from the beads with the addition of 25 U of TEV protease. Eluates were TCA precipitated and analyzed by Western blotting with the indicated antibodies. IP, input; FT, flowthrough; E, eluate.

FIG. 6.

Cdc73-TAP does not associate with components of the proteasome. Extracts from a Cdc73-TAP strain were affinity purified using rabbit IgG agarose beads. Bound proteins were eluted from the resin by adding TEV protease. Eluates were resolved by SDS-PAGE and immunoblotted with antibodies specific to the proteasome. IP, input; FT, flowthrough; E, eluate.

FIG. 7.

A deletion of the RTF1 gene suppresses the defects caused by disruption of the PAF1 gene. (A) Growth curve of indicated deletion strains grown in YPD at 30°C. (B) Cultures of YJJ662 (wild type), YJJ1303 (rtf1Δ::kan^r), YJJ1326 (rtf1Δ::kan^r paf1Δ::HIS3), and YJJ577 (paf1Δ::HIS3) cells (approximately 10⁷/ml) were spotted in 10-fold serial dilutions on YPD medium and grown at 30 and 36°C and on YPD containing 4 mM caffeine and 50 mM hydroxyurea and grown at 30°C. (C) Cultures of YJJ662 (wild type), YJJ1336 (leo1Δ), YJJ1361 (leo1Δ paf1Δ), and YJJ577 (paf1Δ) were grown on the indicated media as described in panel B. Photographs of plates were taken after 4 days of incubation.

FIG. 8.

CLN1 message is restored to wild-type levels in an rtf1Δ paf1Δ double mutant strain. Duplicate cultures of the indicated deletion strains were grown to a density of 1.3 × 10⁷ cells/ml, total RNA was harvested, and 15 μg of RNA per lane was fractionated. mRNA levels were detected using radiolabeled probes to CLN1 and normalized to 18S rRNA as described in Materials and Methods. The bars in the upper panel represent the average of the duplicate samples shown in the lower panel.