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. 2012 Mar 30;287(14):10863-75.
doi: 10.1074/jbc.M111.325647. Epub 2012 Feb 8.

Cdc73 subunit of Paf1 complex contains C-terminal Ras-like domain that promotes association of Paf1 complex with chromatin

Christopher G Amrich  1 , Christopher P Davis, Walter P Rogal, Margaret K Shirra, Annie Heroux, Richard G Gardner, Karen M Arndt, Andrew P VanDemark
Affiliations

Cdc73 subunit of Paf1 complex contains C-terminal Ras-like domain that promotes association of Paf1 complex with chromatin

Christopher G Amrich et al. J Biol Chem. .

Abstract

The conserved Paf1 complex localizes to the coding regions of genes and facilitates multiple processes during transcription elongation, including the regulation of histone modifications. However, the mechanisms that govern Paf1 complex recruitment to active genes are undefined. Here we describe a previously unrecognized domain within the Cdc73 subunit of the Paf1 complex, the Cdc73 C-domain, and demonstrate its importance for Paf1 complex occupancy on transcribed chromatin. Deletion of the C-domain causes phenotypes associated with elongation defects without an apparent loss of complex integrity. Simultaneous mutation of the C-domain and another subunit of the Paf1 complex, Rtf1, causes enhanced mutant phenotypes and loss of histone H3 lysine 36 trimethylation. The crystal structure of the C-domain reveals unexpected similarity to the Ras family of small GTPases. Instead of a deep nucleotide-binding pocket, the C-domain contains a large but comparatively flat surface of highly conserved residues, devoid of ligand. Deletion of the C-domain results in reduced chromatin association for multiple Paf1 complex subunits. We conclude that the Cdc73 C-domain probably constitutes a protein interaction surface that functions with Rtf1 in coupling the Paf1 complex to the RNA polymerase II elongation machinery.

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Figures

FIGURE 1.
FIGURE 1.
Deletion of the Cdc73 C-domain causes phenotypes associated with defects in transcription elongation. A, domain architecture of Cdc73. Sequence conservation is indicated, as are insertions conserved within higher eukaryotes and the locations of known binding interactions for parafibromin. B, Western analysis of tagged Cdc73 proteins. Extracts from strains with the indicated genotypes (KY1021, KY1799, KY1791, KY1802, KY1797, and KY1789) were probed with peroxidase-antiperoxidase antibody to detect the TAP tag. C, dilution analysis assessing the Spt− phenotype of strains with the indicated genotypes (KY1021, KY1857, KY1799, and KY1791). 5-Fold dilutions starting from 1.0 × 108 cells/ml were spotted to SD medium lacking histidine (−His) and incubated at 30 °C for 4 days. D, dilution analyses assessing 6-AU and MPA sensitivity of the strains used in C. 10-Fold dilutions starting from 1.0 × 108 cells/ml were spotted to medium containing 6-AU or MPA and incubated at 30 °C for 4 days.
FIGURE 2.
FIGURE 2.
A C-domain truncation does not prevent Paf1C assembly. A, Western analysis of crude extract and bound fractions from one-step affinity purifications of untagged Cdc73, Cdc73-TAP, and Cdc73ΔC-TAP from strains GHY1185, KY1978, and KY1979. Extracts were probed with indicated antibodies to detect the presence of Paf1C subunits. *, nonspecific band. B, summary of mass spectrometry data. The numbers represent the sums of peptides detected in three independent affinity purifications of untagged Cdc73, Cdc73-TAP, or Cdc73ΔC-TAP from strains KY1021, KY1799, and KY1791. C, Western analysis of Rtf1 levels in bound fractions from one-step affinity purifications using the same strains as in the mass spectrometry analysis.
FIGURE 3.
FIGURE 3.
Structure of the yeast Cdc73 C-domain. A, schematic representation of the yeast Cdc73 C-domain. Elements of secondary structure are indicated and named using the Ras nomenclature. Structural overlap of Cdc73 C-domain (green) and Rab33 (gray) is shown. Bound GTP analog from the Rab33 structure is shown as sticks. The positions of the P-loop (magenta) and the Switch I and II loops (red) important for Ras function are shown. B, primary sequence alignment of S. cerevisiae Cdc73, H-Ras, and Rab33. Above are the secondary structure elements, as determined from the Cdc73 structure. Sequence motifs that are important for Ras function are indicated.
FIGURE 4.
FIGURE 4.
Loops important for nucleotide binding in Ras adopt noncanonical conformations in the Cdc73 C-domain. Structural superposition of the Cdc73 C-domain (green) with Rab33 bound to a GTP analog (Protein Data Bank entry 1Z06). Within the Rab33 structure, the P-loop (magenta), and Switch I and II (red) are indicated, and the position of the GTP analog is shown in sticks. A, loops corresponding to P-loop and Switch I are altered in Cdc73. Residues making important hydrogen bonding interactions with nucleotide and/or Mg ion in Rab33 are indicated as are equivalent residues in Cdc73. B, the Switch II loop region is shifted within Cdc73. Switch II from Rab33 (red) and its interaction with the terminal phosphate is indicated, whereas the position of the equivalent loop in the C-domain is shown in green.
FIGURE 5.
FIGURE 5.
A conserved surface within the Cdc73 C-domain. A, sequence conservation from a multiple sequence alignment of Cdc73 sequences was scored and displayed on the C-domain surface with low conservation in white and invariant residues in red. The residues indicated were mutated and subjected to phenotypic analysis. Residues mutated in HPT-JT patients are indicated by the substitution in Homo sapiens. B, analysis of the 6-AU sensitivity of strains expressing HA-tagged Cdc73 mutant proteins with the indicated amino acid substitutions in the C-domain. Media contained 6-AU and lacked tryptophan for the selection of CEN/ARS plasmid-encoded CDC73 derivatives in strain KY1858. Strains were incubated at 30 °C for 3 days. C, Western analysis of HA-tagged wild-type and mutant Cdc73 proteins. Extracts from the strains analyzed in B were prepared, and Western blots were probed with antibodies specific to the HA tag and glucose-6-phosphate dehydrogenase (G6PDH), which served as a loading control.
FIGURE 6.
FIGURE 6.
Deletion of both the Cdc73 C-domain and Rtf1 leads to enhanced mutant phenotypes and defects in histone modifications. A and C, Western analysis on extracts from strains with the indicated genotypes (KY1021, KY1857, KY1799, KY1791, KY1802, KY703, KY1797, and KY1789) to detect levels of Paf1C-dependent H3 modifications. The arrows indicate the band of interest, and nonspecific bands are marked by an asterisk. B and D, dilution analyses assessing 6-AU and MPA sensitivity of the indicated strains (KY1021, KY1857, KY1802, KY703, KY1797, and KY1789). 10-Fold dilutions starting from 1.0 × 108 cells/ml were spotted to medium containing 6-AU or MPA and incubated at 30 °C for 2 days (D, SC−Ura−Trp), 4 days (B and D, 6-AU), or 8 days (D, MPA). For C and D, cdc73Δ (KY1858) or cdc73Δ rtf1Δ (KY2195) strains were transformed with TRP1-marked CEN/ARS plasmids that express either HA-tagged Cdc73 or HA-tagged Cdc73-W321A. Transformants were grown on media lacking tryptophan.
FIGURE 7.
FIGURE 7.
Deletion of the Cdc73 C-domain reduces Paf1C occupancy on actively transcribed genes. ChIP analysis of HA-tagged Cdc73 (A), Rpb3 (C), Rtf1 (D), and Myc-tagged Ctr9 (E) was measured at the 5′ end of PYK1 (+253 to +346, relative to ATG), 3′ end of PYK1 (+1127 to +1270), 5′ end of PMA1 (+214 to +319), 3′ end of PMA1 (+2107 to +2194) and at an untranscribed region proximal to the telomere of chromosome VI (TEL VI; chromosome coordinates 269,495–269,598). Values represent the average of at least three independent samples with S.D. (error bars). B, Western analysis of HA-tagged Cdc73 levels in strains utilized for ChIP. Extracts from a cdc73Δ strain (KY1858) transformed with empty vector or plasmids expressing full-length untagged Cdc73, full-length HA-tagged Cdc73, or truncated HA-tagged Cdc73ΔC were probed with antibodies specific for the HA tag (Cdc73), Rtf1, or glucose-6-phosphate dehydrogenase (G6PDH) as a loading control. *, nonspecific band. The two bands detected with the Rtf1 antiserum represent full-length Rtf1 and a consistently observed breakdown product.
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