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. 2004 Apr 20;101(16):5904-9.
doi: 10.1073/pnas.0305411101. Epub 2004 Apr 6.

Physical and functional association of RNA polymerase II and the proteasome

Thomas G Gillette  1 , Fernando Gonzalez, Agnes Delahodde, Stephen Albert Johnston, Thomas Kodadek
Affiliations

Physical and functional association of RNA polymerase II and the proteasome

Thomas G Gillette et al. Proc Natl Acad Sci U S A. .

Abstract

Recent studies from a number of laboratories have revealed a surprising number of connections between RNA polymerase II transcription and the ubiquitin/proteasome pathway. We now find yet another intersection of these pathways by showing that the 26S proteasome associates with regions of the GAL1, GAL10, and HSP82 genes, including the 3' ends, in a transcription-dependent fashion. The appearance of the proteasome on these inducible genes correlates with both the accumulation of transcripts and the buildup of RNA polymerase II complexes in the same region. Furthermore, the 26S proteasome and RNA polymerase II coimmunoprecipitate, and inhibition of 26S proteolytic activity leads to increased read through of a transcription termination site. We suggest that the proteasome is generally recruited to the DNA at sites of stalled RNA polymerase and may act to resolve these complexes.

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Figures

Fig. 1.
Fig. 1.
Association of the proteasome with the 3′ end of the GAL1 gene revealed by ChIP analysis. (A) A map of the GAL1 gene showing the regions amplified by PCR. (B) GAL1 PCR products from a ChIP assay. The sample points are in raffinose before GAL gene induction (R), 30 min after galactose addition (G.5), or 2 h after galactose addition (G2) are shown. The numbers correspond to the amount of specific DNA precipitated as a percentage of the input DNA. Totals are 1% of the input DNA.
Fig. 5.
Fig. 5.
Association of the proteasome with the 3′ ends of the GAL10 and HSP82 genes. (A) GAL10 3′ region PCR products from a ChIP assay. Primers are located in a region at the very 3′ end of the GAL10 coding region. The sample points are in raffinose before GAL gene induction (R), 15 min (G.25), 30 min (G.5), 1 h (G1), 2 h (G2), or 4 h (G4) after galactose addition. Totals are 10% of the input DNA (B) HSP82 PCR products from a ChIP assay. The sample points are before heat shock (Un), after heat shock for 15 min (HS), and after removing from heat shock for 20 min (R). Totals are 10% of the input DNA.
Fig. 2.
Fig. 2.
Detailed analysis of the association of Rpb3 and proteasomal proteins with GAL1 by using the ChIP assay. (A) GAL1 PCR products from a ChIP assay. Upstream activation sequence refers to the GAL1–10 promoter region and 3′ to the 3′ region of GAL1 (see Fig. 1A). The sample points are in raffinose before GAL gene induction (R), 30 min (G.5), and 2 h (G2) after galactose addition. Totals are 10% of the input DNA. (B) Graph of the ChIP results. The numbers correspond to the amount of specific DNA precipitated as a percentage of the input DNA. (C) Time course of GAL1 gene induction. GAL1 mRNA levels were normalized to ACT1 as measured by Northern analysis and plotted as a percentage of the peak level over time.
Fig. 3.
Fig. 3.
Inhibition of the 26S proteasome results in increased read through of a termination site. (A) Schematic representation of the reporter constructs. (B) Effect of MG132 on β-gal activity from the reporter constructs. Results of three independent experiments in which strains were induced by galactose for 4 h in the presence or absence of 100 μM MG132, and extracts were obtained. Results are shown as the activity in the untreated sample over the activity of the treated sample.
Fig. 4.
Fig. 4.
Altering the site of termination alters the association of the proteasome with the DNA. (A) Map showing the site of insertion within the GAL1 coding region and location of the primers used in the assay. (B) Northern blot showing the shift in mobility of the GAL1 transcript in the insertion mutant. Sample points are raffinose (R) and 2 h after addition of galactose (G2). Blot was probed with both ACT1 and GAL1 fragments. (C) Association of proteasomal proteins with mutant gal1 strain by using the ChIP assay. Amplified regions are as indicated. The sample points are in raffinose before GAL gene induction (R), 30 min (G.5), and 2 h (G2) after galactose addition. Totals are 10% of the input DNA.
Fig. 6.
Fig. 6.
The proteasome interacts with RNA pol II and associates with transcriptionally active GAL1 after UV irradiation. (A) GAL1 PCR products representing the middle region of the gene from a ChIP assay by using anti-20S antibodies. The sample points are in raffinose (R), raffinose after UV treatment (RUV), 30 min after galactose induction (G), or 30 min after galactose induction and UV treatment (GUV). Totals are 1% of the input DNA. A map of the GAL1 gene shows the region amplified by PCR. (B) Western analysis of immunoprecipitated Rpb3 samples. Extracts from a strain containing a Flag-tagged Rpb3 (T) or an untagged control (C) were immunoprecipitated with anti-Flag antibody and probed for 26S subunits as indicated. Input represents 1% of the total protein added.
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